Nucleic acid detection method

ABSTRACT

The present invention relates to methods for the detection of nucleic acids of defined sequence and kits and devices for use in said methods. The methods employ restriction enzymes, polymerase and oligonucleotide primers to produce an amplification product in the presence of a target nucleic acid, which is contacted with oligonucleotide probes to produce a detector product.

BACKGROUND Technical Field

The present invention is directed to methods for the detection ofnucleic acids of defined sequence and kits and devices for use in saidmethods.

Related Art

Methods of nucleic acid sequence amplification based on polymerases arewidely used in the field of molecular diagnostics. The most establishedmethod, polymerase chain reaction (PCR), typically involves two primersfor each target sequence and uses temperature cycling to achieve primerannealing, extension by DNA polymerase and denaturation of newlysynthesised DNA in a cyclical exponential amplification process. Therequirement for temperature cycling necessitates complex equipment whichlimits the use of PCR-based methods in certain applications.

Strand Displacement Amplification (SDA) (EP0497272; U.S. Pat. Nos.5,455,166; 5,712,124) was developed as an isothermal alternative to PCRthat does not require temperature cycling to achieve the annealing anddenaturation of double stranded DNA during polymerase amplification, andinstead uses restriction enzymes combined with a strand-displacementpolymerase to separate the two DNA strands.

In SDA, a restriction enzyme site at the 5′ end of each primer isintroduced into the amplification product in the presence of one or morealpha thiol nucleotide, and a restriction enzyme is used to nick therestriction sites by virtue of its ability to cleave only the unmodifiedstrand of a hemiphosphorothioate form of its recognition site. A stranddisplacement polymerase extends the 3′-end of each nick and displacesthe downstream DNA strand. Exponential amplification results fromcoupling sense and antisense reactions in which strands displaced from asense reaction serve as target for an antisense reaction and vice versa.SDA typically takes over 1 hour to perform, which has greatly limitedits potential for exploitation in the field of clinical diagnostics.Furthermore, the requirement for separate processes for specificdetection of the product following amplification and to initiate thereaction add significant complexity to the method.

Maples et al. (WO2009/012246) subsequently performed SDA using nickingenzymes, a subclass of restriction enzymes that are only capable ofcleaving one of the two strands of DNA following binding to theirspecific double stranded recognition sequence. They referred to themethod as Nicking and Extension Amplification Reaction (NEAR). NEAR,which employs nicking enzymes instead of restriction enzymes, hassubsequently also been employed by others, who have attempted to improvethe method using software optimised primers (WO2014/164479) and througha warm start or controlled reduction in temperature (WO2018/002649).However, only a very small number of nicking enzymes are available andthus it is more challenging to find an enzyme with the desiredproperties for a particular application.

A crucial disadvantage of SDA using either restriction enzymes ornicking enzymes (NEAR) is that it produces a double stranded nucleicacid product and thus does not provide an intrinsic process forefficient detection of the amplification signal. This has significantlylimited its utility in, for example, low-cost diagnostic devices. Thedouble stranded nature of the amplified product produced presents achallenge for coupling the amplification method to signal detectionsince it is not possible to perform hybridisation-based detectionwithout first separating the two strands. Therefore more complexdetection methods are required, such as molecular beacons andfluorophore/quencher probes, which can complicate assay protocols byrequiring a separate process step and significantly reduces thepotential to develop multiplex assays.

There is an important requirement for enhanced amplification methods forrapid, sensitive and specific nucleic acid sequence detection toovercome the limitations of SDA. The present invention relates to amethod of target nucleic acid sequence amplification and detectionwhich, in addition to a pair of primers with 5′ restriction sites,utilises additional oligonucleotide probes to produce a detector speciesthat enables efficient signal detection.

SUMMARY

The invention provides a method for detecting the presence of a singlestranded target nucleic acid of defined sequence in a sample comprising:

-   -   a) contacting the sample with:        -   i. a first oligonucleotide primer and a second            oligonucleotide primer wherein said first primer comprises            in the 5′ to 3′ direction one strand of a restriction enzyme            recognition sequence and cleavage site and a region that is            capable of hybridising to a first hybridisation sequence in            the target nucleic acid, and said second primer comprises in            the 5′ to 3′ direction one strand of a restriction enzyme            recognition sequence and cleavage site and a region that is            capable of hybridising to the reverse complement of a second            hybridisation sequence upstream of the first hybridisation            sequence in the target nucleic acid;        -   ii. a strand displacement DNA polymerase;        -   iii. dNTPs;        -   iv. one or more modified dNTP;        -   v. a first restriction enzyme that is not a nicking enzyme            but is capable of recognising the recognition sequence of            the first primer and cleaving only the first primer strand            of the cleavage site when said recognition sequence and            cleavage site are double stranded, the cleavage of the            reverse complementary strand being blocked due to the            presence of one or more modifications incorporated into said            reverse complementary strand by the DNA polymerase using the            one or more modified dNTP; and        -   vi. a second restriction enzyme that is not a nicking enzyme            but is capable of recognising the recognition sequence of            the second primer and cleaving only the second primer strand            of the cleavage site when said recognition sequence and            cleavage site are double stranded, the cleavage of the            reverse complementary strand being blocked due to the            presence of one or more modifications incorporated into said            reverse complementary strand by the DNA polymerase using the            one or more modified dNTP;        -   to produce, without temperature cycling, in the presence of            said target nucleic acid, amplification product;    -   b) contacting the amplification product of step a) with:        -   i. a first oligonucleotide probe which is capable of            hybridising to a first single stranded detection sequence in            at least one species within the amplification product and            which is attached to a moiety that permits its detection;            and        -   ii. a second oligonucleotide probe which is capable of            hybridising to a second single stranded detection sequence            upstream or downstream of the first single stranded            detection sequence in said at least one species within the            amplification product and which is attached to a solid            material or to a moiety that permits its attachment to a            solid material;        -   where hybridisation of the first and second probes to said            at least one species within the amplification product            produces a detector species; and    -   c) detecting the presence of the detector species produced in        step b) wherein the presence of the detector species indicates        the presence of the target nucleic acid in said sample.

An embodiment of the method is illustrated in FIG. 1.

In various embodiments, in the presence of target nucleic acid, themethod rapidly produces many copies of the detector species which isideally suited to sensitive detection.

The present invention in various aspects is advantageous over knownmethods because it encompasses rapid amplification without temperaturecycling in addition to providing an intrinsic process for efficientdetection of the amplified product.

The method of the invention overcomes a major disadvantage of SDA,including SDA with nicking enzymes (NEAR), which is that SDA does notprovide an intrinsic process for efficient detection of theamplification signal due to the double stranded nature of theamplification product. The present method overcomes this limitation byutilising two additional oligonucleotide probes which hybridise to atleast one species in the amplification product to facilitate its rapidand specific detection. The use of these two additional oligonucleotideprobes, the first of which is attached to a moiety that permits itsdetection and the second of which is attached to a solid material or amoiety that permits it attachment to a solid material, provide a numberof further advantages to the present invention over known methods suchas SDA. For example, in embodiments of the invention wherein one of theoligonucleotide probes is blocked at the 3′ end from extension by theDNA polymerase, is not capable of being cleaved by the restrictionenzyme(s) and is contacted with the sample simultaneously to theperformance of step a), surprisingly no significant detrimentalinhibition of the amplification is observed and a pre-detector speciescontaining a single stranded region is produced efficiently. This aspectof the invention is counter-intuitive as it may be assumed that such ablocked probe would lead to asymmetric amplification that is biased tothe opposite amplification product strand to that comprised in thepre-detector species. In fact, said pre-detector species is efficientlyproduced and ideally suited to efficient detection because the exposedsingle stranded region is readily available for hybridisation of theother oligonucleotide probe.

The intrinsic sample detection approach of the present method contrastsfundamentally with prior attempts to overcome this important limitationof SDA which involved performing “asymmetric” amplification, forexample, by using an unequal primer ratio with a goal of producing anexcess of one amplicon strand over the other. The present method doesnot require asymmetric amplification nor does it have any requirement toproduce an excess of one strand of the amplicon over the other andinstead it is focussed on production of the detector species followinghybridisation of the first and second oligonucleotide probes. Theintrinsic sample detection approach of the present method involvingproduction of a detector species is ideally suited to its coupling with,amongst other detection methods, nucleic acid lateral flow, providing asimple, rapid and low-cost means of performing detection in step c), forexample, by printing the second oligonucleotide probe on the lateralflow strip. When coupled to nucleic acid lateral flow the method alsopermits efficient multiplexing based upon differential hybridisation ofmultiple second oligonucleotide probes attached at discrete locations onthe lateral flow strip, each with a different sequence designed for adifferent target nucleic acid sequence in the sample. In furtherembodiments of the method, the efficiency of the lateral flow detectionis enhanced by the use of a single stranded oligonucleotide as themoiety within the second oligonucleotide probe that permits itsattachment to a solid material, and the reverse complementary sequenceto said moiety is printed on the strip. The latter approach also permitsthe lateral flow strip to be optimised and manufactured as a single“universal” detection system across multiple target applications becausethe sequences attached to the lateral flow strip can be defined and donot need to correspond to the sequence of the target nucleic acid(s).The integral requirement for two additional oligonucleotide probes inthe method of the invention thus provides many advantages over SDA,including SDA with nicking enzymes (NEAR).

Since the present invention requires the use of restriction enzyme(s)that are not nicking enzymes and one or more modified dNTP, it isfundamentally different to SDA performed using nicking enzymes (NEAR)and has a number of further advantages over such nicking enzymedependent methods. For example, a much greater number of restrictionenzymes that are not nicking enzymes are available than nicking enzymes,which means that the restriction enzyme(s) for use in the method of theinvention can be selected from a large number of potential enzymes toidentify those with superior properties for a given application, e.g.reaction temperature, buffer compatibility, stability and reaction rate(sensitivity). Due to this key advantage of the present method, we havebeen able to select restriction enzymes with a lower temperature optimumand a faster rate than would be possible to achieve with nickingenzymes. Such restriction enzymes are much better suited to exploitationin a low-cost diagnostic device. Furthermore the requirement to use oneor more modified dNTP is an integral feature of the present inventionwhich offers important advantages in addition to providing for therestriction enzymes to cleave only one strand of their restrictionsites. For example, certain modified dNTPs, such as alpha thiol dNTPs,lead to a reduction in the melting temperature (Tm) of the DNA intowhich they are incorporated which means the oligonucleotide primers andprobes in the method have a greater affinity for hybridisation to thespecies within the amplification product than any competingcomplementary strand containing modified dNTP produced during theamplification. Furthermore, the reduction in Tm of the amplificationproduct as a result of modified dNTP base insertion facilitates theseparation of double stranded DNA species and thus enhances the rate ofamplification, reduces the temperature optimum and improves thesensitivity. Alternatively, other modified dNTPs can increase the Tm ofthe DNA into which they are incorporated presenting furtheropportunities to tailor the performance of the method for a givenapplication.

Together the numerous advantages of the present invention over SDA,using either restriction enzymes or nicking enzymes (NEAR), provide forthe utility of the method in low-cost, single-use diagnostic devices, byvirtue of the improved rate of amplification and simple visualisation ofthe amplification signal that are not possible with known methods.

Various embodiments of the above mentioned aspects of the invention, andfurther aspects, are described in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of the method according to one aspectof the invention.

FIG. 2. Schematic representation of the method wherein the firstoligonucleotide probe is blocked at the 3′ end from extension by the DNApolymerase and is not capable of being cleaved by either the first orsecond restriction enzyme and is contacted with the sample in step a).

FIG. 3. Schematic representation of steps b) and c) of the methodwherein the moiety that permits the attachment of the secondoligonucleotide probe to a solid material is a single strandedoligonucleotide.

FIG. 4. Schematic representation of part of step a) of the methodwherein the sample is additionally contacted with a third and fourtholigonucleotide primer in step a).

FIG. 5. Performance of the method wherein the second oligonucleotideprobe is attached to a solid material, a nitrocellulose lateral flowstrip (see Example 1).

FIGS. 6A and 6B. Performance of the method wherein the firstoligonucleotide probe is blocked at the 3′ end from extension by the DNApolymerase and is not capable of being cleaved by either the first orsecond restriction enzyme and is contacted with the sample in step a)(see Example 2).

FIGS. 7A, 7B, 7C and 7D. Performance of the method wherein the presenceof two of more different target nucleic acids of defined sequence aredetected in the same sample (see Example 3).

FIG. 8. Performance of the method wherein the first and secondhybridisation sequences in the target nucleic acid are separated by 5bases (see Example 4).

FIG. 9. Performance of the method wherein the moiety that permits theattachment of the second oligonucleotide probe to a solid material is anantigen and the corresponding antibody is attached to a solid surface, anitrocellulose lateral flow strip (see Example 5).

FIGS. 10A and 10B. Performance of the method wherein the moiety thatpermits the attachment of the second oligonucleotide probe to a solidmaterial is a single stranded oligonucleotide comprising four repeatcopies of a three base DNA sequence motif and the reverse complement ofsaid single stranded oligonucleotide sequence is attached to a solidmaterial (see Example 6).

FIG. 11. Use of the method for the detection of an RNA virus in clinicalspecimens (see Example 7).

FIGS. 12A and 12B. Performance of the method at different temperatures(see Example 8).

FIGS. 13A and 13B. Performance of the method wherein the target nucleicacid is derived from double stranded DNA by strand invasion (see Example9).

FIGS. 14A and 14B. Comparative performance of the method of theinvention versus known methods (see Example 10).

DETAILED DESCRIPTION

The present invention provides a method for detecting the presence of asingle stranded target nucleic acid of defined sequence in a sample. Thetarget nucleic acid may be single stranded DNA, including singlestranded DNA derived from double stranded DNA following disassociationof the two strands in the sample such as by heat denaturation or throughstrand displacement activity of a polymerase, or derived from RNA e.g.by the action of reverse transcriptase, or derived from double strandedDNA e.g. by use of a nuclease, such as a restriction endonuclease orexonuclease III, or derived from a RNA/DNA hybrid e.g. through an enzymesuch as Ribonuclease H. The target nucleic acid may be single strandedDNA derived from DNA in the sample by a DNA polymerase, helicase orrecombinase. Single stranded sites within double stranded DNA may beexposed sufficiently for hybridisation and extension of the firstoligonucleotide primer to initiate the method, for example by “strandinvasion” wherein transient opening of one or more DNA base pairs withinthe double stranded DNA occurs sufficiently to permit hybridisation andextension of the 3′ hydroxyl of the first oligonucleotide primer, or byspontaneous opening of DNA base pairs, transient conversion to Hoogsteenpairs or productive nicking of DNA by restriction enzyme orthermochemical approaches. The target nucleic acid may be singlestranded RNA, including single stranded RNA derived from double strandedRNA in the sample following disassociation of the two strands such as byheat denaturation or single stranded RNA derived from double strandedDNA e.g. by transcription.

The method involves in step a) contacting the sample with: (i) a firstoligonucleotide primer and a second oligonucleotide primer wherein saidfirst primer comprises in the 5′ to 3′ direction one strand of arestriction enzyme recognition sequence and cleavage site and a regionthat is capable of hybridising to a first hybridisation sequence in thetarget nucleic acid, and said second primer comprises in the 5′ to 3′direction one strand of a restriction enzyme recognition sequence andcleavage site and a region that is capable of hybridising to the reversecomplement of a second hybridisation sequence upstream of the firsthybridisation sequence in the target nucleic acid; (ii) a stranddisplacement DNA polymerase; (iii) dNTPs; (iv) one or more modifieddNTP; (v) a first restriction enzyme that is not a nicking enzyme but iscapable of recognising the recognition sequence of the first primer andcleaving only the first primer strand of the cleavage site when saidrecognition sequence and cleavage site are double stranded, the cleavageof the reverse complementary strand being blocked due to the presence ofone or more modifications incorporated into said reverse complementarystrand by the DNA polymerase using the one or more modified dNTP; and(vi) a second restriction enzyme that is not a nicking enzyme but iscapable of recognising the recognition sequence of the second primer andcleaving only the second primer strand of the cleavage site when saidrecognition sequence and cleavage site are double stranded, the cleavageof the reverse complementary strand being blocked due to the presence ofone or more modifications incorporated into said reverse complementarystrand by the DNA polymerase using the one or more modified dNTP.

When the target nucleic acid to be detected in the sample is doublestranded either strand may be deemed the single stranded target nucleicacid of the method since one of the two oligonucleotide primers iscapable of hybridisation to one strand and the other oligonucleotideprimer is capable of hybridisation to the other strand. Typically, theoligonucleotide primers used in the method are DNA primers which formwith the DNA or RNA target a double stranded DNA or a hybrid duplexcomprising strands of both RNA and DNA. However, primers comprisingother nucleic acids, such as non-natural bases and/or alternativebackbone structures, may also be used.

In the presence of the target nucleic acid the first oligonucleotideprimer hybridises to the first hybridisation sequence in the targetnucleic acid. Following said hybridisation, the 3′ hydroxyl group of thefirst primer is extended by the strand displacement DNA polymerase or,optionally, in the case of an RNA target nucleic acid a reversetranscriptase (e.g. M-MuLV), to produce a double stranded speciescontaining the extended first primer and the target nucleic acid (seeFIG. 1). The strand displacement DNA polymerase or, when present, thereverse transcriptase use the dNTPs and the one or more modified dNTP insaid extension. The one strand of a restriction enzyme recognitionsequence and cleavage site at the 5′ end of the first primer does nottypically hybridise as the reverse complementary sequence thereto isgenerally not present in the target nucleic acid sequence. Thus thefirst primer is generally used to introduce said one strand of arestriction enzyme recognition sequence and cleavage site intosubsequent amplification product species. Following extension of thefirst primer, “target removal” occurs. Target removal makes accessiblethe extended first primer species for hybridisation of the secondoligonucleotide primer to the reverse complement of the secondhybridisation sequence. When the target nucleic acid is RNA, targetremoval may be accomplished, for example, by RNase H degradation of theRNA, accomplished through the RNase H activity of the reversetranscriptase if present or through separate addition of this enzyme.Alternatively, when the target nucleic acid is single stranded DNA,including a single-stranded region within double stranded DNA, it may beaccomplished by strand displacement using an additional upstream primeror bump primer. Alternatively, such target removal may occur followingspontaneous disassociation, particularly if only a short extensionproduct has been produced from a given target nucleic acid molecule, orit may occur through strand invasion wherein transient opening of one ormore DNA base pairs within the double stranded extended first primerspecies occurs sufficiently to permit hybridisation and extension of the3′ hydroxyl of the second oligonucleotide primer with stranddisplacement.

Following hybridisation of the second oligonucleotide primer to thereverse complement of the second hybridisation sequence, the stranddisplacement DNA polymerase extends the 3′ hydroxyl of said primer usingthe dNTPs and the one or more modified dNTP. The double strandedrestriction recognition sequence and cleavage site for the firstrestriction enzyme is formed with one or more modified dNTP base(s)incorporated into the reverse complementary strand acting to block thecleavage of said strand by said first restriction enzyme. The firstrestriction enzyme recognises its recognition sequence and cleaves onlythe first primer strand of the cleavage site, creating a 3′ hydroxylthat is extended by the strand displacement DNA polymerase using thedNTPs and the one or more modified dNTP and displacing the first primerstrand. The double stranded restriction recognition sequence andcleavage site for the second restriction enzyme is formed with one ormore modified dNTP base(s) incorporated into the reverse complementarystrand acting to block the cleavage of said strand by said secondrestriction enzyme. A double stranded species is thus produced in whichthe two primer sequences are juxtaposed and the partially blockedrestriction site of the first restriction enzyme and second restrictionenzyme are present. The cleavage by the first restriction enzyme of thefirst primer strand and by the second restriction enzyme of the secondprimer strand then occur, and two double stranded species are produced,one comprising the first primer sequence and the other comprising asecond primer sequence. The sequential cleavage and displacement of thefirst primer strand and the second primer strand then occur in acyclical amplification process wherein the displaced first primer strandacts as a target for the second primer and the displaced second primerstrand acts as a target for the first primer.

In the presence of target nucleic acid, amplification product isproduced without any requirement for temperature cycling.

An integral aspect of the present invention is that rather than directdetection of the amplification product of step a), a detector species isproduced following the specific hybridisation of both a first and asecond oligonucleotide probe to at least one species within theamplification product. The first oligonucleotide probe, which isattached to a moiety that permits its detection, hybridises to a firstsingle stranded detection sequence in said at least one species. Thesecond oligonucleotide probe, which is attached to a solid material orto a moiety that permits its attachment to a solid material, hybridisesto a second single stranded detection sequence upstream or downstream ofthe first single stranded detection sequence in said at least onespecies.

It will be apparent to a skilled person, with reference to FIG. 1, thatamplification product comprises a number of different species, such asspecies comprising single stranded detection sequences, consisting ofthe full or partial sequence or reverse complementary sequence of boththe first primer and second primer, which sequences may be separated bytarget-derived sequence in the event that the primer binding first andsecond hybridisation sequences in the target nucleic acid are separatedby one or more bases. It will further be apparent that any of saidspecies may be selected to hybridise to the first and secondoligonucleotide probe to form the detector species.

The detector species produced in step b) is detected in step c), whereinthe presence of the detector species indicates the presence of thetarget nucleic acid in the sample.

By utilising two oligonucleotide probes, one for detection and one forattachment to a solid material, the method of the invention provides forrapid and efficient signal detection, which overcomes the requirementfor more complex secondary detection methods and provides for efficientvisualisation of the signal produced in the presence of target, such asby nucleic acid lateral flow.

The method of the invention may be performed wherein one of the firstand second oligonucleotide probes is blocked at the 3′ end fromextension by the strand displacement DNA polymerase and is not capableof being cleaved by either the first or second restriction enzymes. Thusaccording to a further embodiment the invention provides a method fordetecting the presence of a single stranded target nucleic acid ofdefined sequence in a sample comprising:

-   -   a) contacting the sample with:        -   i. a first oligonucleotide primer and a second            oligonucleotide primer wherein said first primer comprises            in the 5′ to 3′ direction one strand of a restriction enzyme            recognition sequence and cleavage site and a region that is            capable of hybridising to a first hybridisation sequence in            the target nucleic acid, and said second primer comprises in            the 5′ to 3′ direction one strand of a restriction enzyme            recognition sequence and cleavage site and a region that is            capable of hybridising to the reverse complement of a second            hybridisation sequence upstream of the first hybridisation            sequence in the target nucleic acid;        -   ii. a strand displacement DNA polymerase;        -   iii. dNTPs;        -   iv. one or more modified dNTP;        -   v. a first restriction enzyme that is not a nicking enzyme            but is capable of recognising the recognition sequence of            the first primer and cleaving only the first primer strand            of the cleavage site when said recognition sequence and            cleavage site are double stranded, the cleavage of the            reverse complementary strand being blocked due to the            presence of one or more modifications incorporated into said            reverse complementary strand by the DNA polymerase using the            one or more modified dNTP; and        -   vi. a second restriction enzyme that is not a nicking enzyme            but is capable of recognising the recognition sequence of            the second primer and cleaving only the second primer strand            of the cleavage site when said recognition sequence and            cleavage site are double stranded, the cleavage of the            reverse complementary strand being blocked due to the            presence of one or more modifications incorporated into said            reverse complementary strand by the DNA polymerase using the            one or more modified dNTP;        -   to produce, without temperature cycling, in the presence of            said target nucleic acid, amplification product;    -   b) contacting the amplification product of step a) with:        -   i. a first oligonucleotide probe which is capable of            hybridising to a first single stranded detection sequence in            at least one species within the amplification product and            which is attached to a moiety that permits its detection;            and        -   ii. a second oligonucleotide probe which is capable of            hybridising to a second single stranded detection sequence            upstream or downstream of the first single stranded            detection sequence in said at least one species within the            amplification product and which is attached to a solid            material or to a moiety that permits its attachment to a            solid material;        -   wherein one of the first and second oligonucleotide probes            is blocked at the 3′ end from extension by the DNA            polymerase and is not capable of being cleaved by either the            first or second restriction enzymes, and where hybridisation            of the first and second probes to said at least one species            within the amplification product produces a detector            species; and    -   c) detecting the presence of the detector species produced in        step b) wherein the presence of the detector species indicates        the presence of the target nucleic acid in said sample.

In an embodiment said one blocked oligonucleotide probe is rendered notcapable of being cleaved by either the first or second restrictionenzymes due to the presence of one or more sequence mismatch and/or oneor more modifications such as a phosphorothioate linkage. In a furtherembodiment the one blocked oligonucleotide probe is contacted with thesample simultaneously to the performance of step a), i.e. during theperformance of step a) such that it is present during the production ofamplification product in the presence of the target nucleic acid. Thusaccording to a further embodiment the invention provides a method fordetecting the presence of a single stranded target nucleic acid ofdefined sequence in a sample comprising:

-   -   a) contacting the sample with:        -   i. a first oligonucleotide primer and a second            oligonucleotide primer wherein said first primer comprises            in the 5′ to 3′ direction one strand of a restriction enzyme            recognition sequence and cleavage site and a region that is            capable of hybridising to a first hybridisation sequence in            the target nucleic acid, and said second primer comprises in            the 5′ to 3′ direction one strand of a restriction enzyme            recognition sequence and cleavage site and a region that is            capable of hybridising to the reverse complement of a second            hybridisation sequence upstream of the first hybridisation            sequence in the target nucleic acid;        -   ii. a strand displacement DNA polymerase;        -   iii. dNTPs;        -   iv. one or more modified dNTP;        -   v. a first restriction enzyme that is not a nicking enzyme            but is capable of recognising the recognition sequence of            the first primer and cleaving only the first primer strand            of the cleavage site when said recognition sequence and            cleavage site are double stranded, the cleavage of the            reverse complementary strand being blocked due to the            presence of one or more modifications incorporated into said            reverse complementary strand by the DNA polymerase using the            one or more modified dNTP; and        -   vi. a second restriction enzyme that is not a nicking enzyme            but is capable of recognising the recognition sequence of            the second primer and cleaving only the second primer strand            of the cleavage site when said recognition sequence and            cleavage site are double stranded, the cleavage of the            reverse complementary strand being blocked due to the            presence of one or more modifications incorporated into said            reverse complementary strand by the DNA polymerase using the            one or more modified dNTP;        -   to produce, without temperature cycling, in the presence of            said target nucleic acid, amplification product;    -   b) contacting the amplification product of step a) with:        -   i. a first oligonucleotide probe which is capable of            hybridising to a first single stranded detection sequence in            at least one species within the amplification product and            which is attached to a moiety that permits its detection;            and        -   ii. a second oligonucleotide probe which is capable of            hybridising to a second single stranded detection sequence            upstream or downstream of the first single stranded            detection sequence in said at least one species within the            amplification product and which is attached to a solid            material or to a moiety that permits its attachment to a            solid material;        -   wherein one of the first and second oligonucleotide probes            is blocked at the 3′ end from extension by the DNA            polymerase, is not capable of being cleaved by either the            first or second restriction enzymes and is contacted with            the sample simultaneously to the performance of step a), and            where hybridisation of the first and second probes to said            at least one species within the amplification product            produces a detector species; and    -   c) detecting the presence of the detector species produced in        step b) wherein the presence of the detector species indicates        the presence of the target nucleic acid in said sample.

For example, in the embodiment illustrated in FIG. 2, the firstoligonucleotide probe is blocked and hybridises to the first singlestranded detection sequence in at least one species within theamplification product to form a pre-detector species containing a singlestranded region. Said at least one species may be extended by the stranddisplacement DNA polymerase extending its 3′ hydroxyl group and thusfurther stabilising said pre-detector species. Thus, in said embodimentthe blocked oligonucleotide probe comprises an additional region suchthat the 3′ end of the species within the amplification product to whichthe blocked oligonucleotide probe hybridises can be extended by thestrand displacement DNA polymerase. A “Stabilised Pre-detector Species”is produced as displayed in FIG. 2. The skilled person will appreciatethat this additional pre-detector species stabilisation region in theblocked oligonucleotide probe will be upstream of the region thathybridises to either the first or second single stranded detectionsequence in the at least one species within the amplification product Inembodiments using a blocked oligonucleotide probe the hybridisationsequence of the blocked oligonucleotide probe and the relevantconcentrations of the primers may be optimised such that a certainproportion of the relevant species produced in the amplification producthybridises to the blocked oligonucleotide probe in each cycle and theremaining copies of such species remain available to participate in thecyclical amplification process. The oligonucleotide probe is blockedfrom extension, for example, by use of a 3′ phosphate modification and,in this embodiment, is also attached to a moiety that permits itsdetection, such as a 5′ biotin modification. Alternatively a single 3′modification may be used to block extension and as a moiety that permitsits detection. Various other modifications are available to block the 3′end of oligonucleotides such as a C-3 spacer; alternatively mismatchbase(s) may be employed. Said pre-detector species is ideally suited toefficient detection because the exposed single stranded region remainsreadily available for hybridisation to the second oligonucleotide probe.The second oligonucleotide probe may be attached to the nitrocellulosesurface of a nucleic acid lateral flow strip such that when thepre-detector species flows over it sequence specific hybridisationreadily occurs and the detector species becomes located at a definedlocation on the strip. A dye which attaches to the detection moiety,such as a streptavidin attached carbon, gold or polystyrene particle,that may be present in the conjugate pad of the nucleic acid lateralflow strip or during the amplification reaction, provides a rapidcolour-based visualisation of the presence of the detector speciesproduced in the presence of the target nucleic acid.

In another embodiment it is the second oligonucleotide probe that isblocked at the 3′ end from extension by the strand displacement DNApolymerase and is not capable of being cleaved by either the first orsecond restriction enzymes and is contacted with the samplesimultaneously to the performance of step a). The second oligonucleotideprobe may be attached to a solid material, such as the surface of anelectrochemical probe, 96-well plate, beads or array surface, prior tobeing contacted with the sample, or may be attached to a moiety thatpermits its attachment to a solid material. A certain proportion of atleast one species produced during the amplification hybridises to thesecond oligonucleotide probe following its production, instead ofhybridising to the relevant reaction primer to participate further inthe cyclical amplification process. Following hybridisation to thesecond oligonucleotide probe, said species are extended by thepolymerase onto the oligonucleotide probe to produce the stabilisedpre-detector species. The first oligonucleotide probe and detectionmoiety may also be contacted with the sample simultaneously to theperformance of step a) and would become localised to said surface at thesite of the second oligonucleotide probe. By detecting the accumulationof the detection moiety at the site during the amplification process areal-time signal would be obtained providing for a quantitation of thenumber of copies of target nucleic acid present in the sample. Thusaccording to an embodiment of the invention, two or more of steps a), b)and c) are performed simultaneously.

In the performance of those embodiments wherein one of the first andsecond oligonucleotide probes is blocked at the 3′ end from extension bythe DNA polymerase and is not capable of being cleaved by either thefirst or second restriction enzymes and is contacted with the samplesimultaneously to the performance of step a), we have not observed anysignificant inhibition of the rate of the amplification, indicating thatthe pre-detector species accumulates in real-time without disrupting theoptimal cyclical amplification process. This contrasts with attempts toengineer asymmetric SDA by utilising an unequal primer ratio with thegoal of producing an excess of one amplicon strand over the other.Rather than seeking to use the blocked oligonucleotide probe to removeone amplicon strand from the reaction and thus increase the proportionof the other strand, the present invention is focussed on the productionand detection of the detector species exploiting a blocked probe tofacilitate the exposure of a single stranded region during theamplification process. Thus not only did we not observe any inhibitoryeffects on the amplification process in said embodiments but we observeda surprising enhancement of the signal produced corresponding to anincreased amount of detector species, of at least 100-fold in certainembodiments, see Example 2 (FIG. 6).

Further, said embodiments of the method of the invention wherein one ofthe first and second oligonucleotides probes is blocked at the 3′ endfrom extension by the DNA polymerase and is not capable of being cleavedby either the first or second restriction enzymes and is contacted withthe sample simultaneously to the performance of step a), represent afundamental advantage over reported attempts to integrate NEAR withnucleic acid lateral flow in a multistep process without blocked probes.For example, in WO2014/164479 a long incubation of 30 minutes at 48° C.was required to visualise amplification product using nucleic acidlateral flow, which represents a major impediment to the use of thatmethod in a point-of-care diagnostic device, particularly a low-cost orsingle-use device. In stark contrast, the method of the inventionreadily performs an equivalent amplification in under 5 minutes and at alower temperature of incubation, e.g. 40-45° C. In a further directcomparative study (see Example 10), the method of the inventiondemonstrates a surprising vastly superior rate compared to a the priorart method (WO2014/164479) resulting from a combination of the use of arestriction enzyme that is not a nicking enzyme, the use of a modifieddNTP base and the use of said blocked oligonucleotide probe.

It will also be appreciated that the other of the first and secondoligonucleotide probes may be blocked at the 3′ end from extension bythe DNA polymerase, and/or is not capable of being cleaved by either thefirst or second restriction enzymes, as described above.

An integral aspect of the method is the use of one or more restrictionenzyme that is not a nicking enzyme, but is capable of recognising itsrecognition sequence and cleaving only one strand of its cleavage sitewhen said recognition sequence and cleavage site are double stranded,the cleavage of the reverse complementary strand being blocked due tothe presence of one or more modifications incorporated into said reversecomplementary strand by a strand displacement DNA polymerase using oneor more modified dNTP, e.g. a dNTP that confers nuclease resistancefollowing its incorporation by a polymerase.

A “restriction enzyme” [or “restriction endonuclease”] is a broad classof enzyme which cleaves one or more phosphodiester bond on one or bothstrands of a double stranded nucleic acid molecule at specific cleavagesites following binding to a specific recognition sequence. A largenumber of restriction enzymes are available, with over 3,000 reportedand over 600 commercially available, covering a wide range of differentphysicochemical properties and recognition sequence specificities.

A “nicking enzyme” [or “nicking endonuclease”] is a particular subclassof restriction enzyme, that is only capable of cleaving one strand of adouble stranded nucleic acid molecule at a specific cleavage sitefollowing binding to a specific recognition sequence, leaving the otherstrand intact. Only a very small number (c.10) nicking enzymes areavailable including both naturally occurring and engineered enzymes.Nicking enzymes include bottom strand cutters Nb.BbvCI, Nb.BsmI,Nb.BsrDI, Nb.BssSI and Nb.BtsI and top strand cutters Nt.AlwI, Nt.BbvCI,Nt.BsmAI, Nt.BspQI, Nt.BstNBI and Nt.CviPII.

Restriction enzymes that are not nicking enzymes, which are exclusivelyemployed in the method of the invention, despite being capable ofcleaving both strands of a double stranded nucleic acid, can in certaincircumstances also cleave or nick only one strand of their doublestranded DNA cleavage site following binding to their recognitionsequence. This can be accomplished in a number of ways. Of particularrelevance to the present method this can be accomplished when one of thestrands within the double stranded nucleic acid at the cleavage site isrendered not capable of being cleaved due to one strand of the doublestranded nucleic acid target site being modified such that thephosphodiester bond of the cleavage site on one of the strands isprotected using a nuclease resistant modification, such as aphosphorothioate (PTO), boranophosphate, methylphosphate or peptideinternucleotide linkage. Certain modified internucleotide linkages, e.g.PTO linkages, can be chemically synthesised within oligonucleotidesprobes and primers or integrated into a double stranded nucleic acid bya polymerase, such as by using one or more alpha thiol modifieddeoxynucleotide. Thus, in an embodiment the one or more modified dNTP isan alpha thiol modified dNTP. Typically the S isomer is employed whichis incorporated and confers nuclease resistance more effectively.

Due to the very large number of restriction enzymes that are not nickingenzymes available, a wide range of enzymes with different properties areavailable to be screened for the desired performance characteristics,e.g. temperature profile, rate, buffer compatibility, polymerasecross-compatibility, recognition sequence, thermostability,manufacturability etc., for use in the method for a given application.In contrast the fact that only a small number of nicking enzymes areavailable limits the potential of prior art methods that use nickingenzymes, and can lead to a lower reaction rate (sensitivity, time toresult) and a higher reaction temperature, for example. Restrictionenzymes that are not nicking enzymes selected for use in the method maybe naturally occurring or engineered enzymes.

In selecting the restriction enzyme that is not a nicking enzyme for usein the method the skilled person will recognise that it is necessary toidentify an enzyme with an appropriate cleavage site in order to ensurethat a modification is incorporated at the correct position to block thecleavage of the relevant strand and not the other strand. For example,in an embodiment in which a modified dNTP, such as an alpha thiol dNTP,is used it may be preferable to select a restriction enzyme with acleavage site that falls outside of the recognition sequence, such as anasymmetric restriction enzyme with a non-palindromic recognitionsequence, in order to provide sufficient flexibility to position theprimers such that the target nucleic acid sequence contains the modifiednucleotide base at the appropriate location to block the cleavage of therelevant strand following its incorporation. For example, if alpha thioldATP is used the reverse complementary sequence of the restrictionenzyme cleavage site in the relevant oligonucleotide primer wouldcontain an Adenosine base downstream of the cleavage position in saidreverse complementary strand but not contain an Adenosine basedownstream of the cleavage site in the primer sequence, in order toensure that primer is cleaved appropriately in the performance of themethod. Therefore asymmetric restriction enzymes with a non-palindromicrecognition sequence that cleave outside of their recognition sequenceare ideally suited for use in the present invention. Partial ordegenerate palindromic sequence recognising restriction enzymes thatcleave within their recognition site may also be used. Nucleaseresistant nucleotide linkage modifications, e.g. PTO, may be used toblock the cleavage of either strand by a wide range of commerciallyavailable double strand cleaving agents of various different classes,including type IIS and type IIG restriction enzymes with both partial ordegenerate palindromic and asymmetric restriction recognition sequences,in order to enable their use in the method of the invention.

Restriction enzyme(s) are typically employed in the method in an amountof 0.1-100 Units, where one unit is defined as the amount of agentrequired to digest 1 μg T7 DNA in 1 hour at a given temperature (e.g.37° C.) in a total reaction volume of 50 μl. However, the amount dependson a number of factors such as the activity of the enzyme selected, theconcentration and form of the enzyme, the anticipated concentration ofthe target nucleic acid, the volume of the reaction, the concentrationof the primers and the reaction temperature, and should not beconsidered limiting in any way. Those skilled in the art will understandthat a restriction enzyme employed in the method will require a suitablebuffer and salts, e.g. divalent metal ions, for effective and efficientfunction, control of pH and stabilisation of the enzyme.

In an embodiment the first and second restriction enzyme are the samerestriction enzyme.

By using only a single restriction enzyme the method is simplified in anumber of ways. For example, only a single enzyme that is compatiblewith other reaction components needs to be identified, optimised forperformance of the method, manufactured and stabilised. Utilising asingle restriction enzyme also simplifies design of oligonucleotideprimers and supports the symmetry of the amplification process.

In the method the restriction enzymes cleave only one strand of thenucleic acid duplex, and thus following cleavage they present an exposed3′ hydroxyl group which can act as an efficient priming site for apolymerase. A polymerase is an enzyme that synthesises chains orpolymers of nucleic acids by extending a primer and generating a reversecomplementary “copy” of a DNA or RNA template strand using base-pairinginteractions. A polymerase with strand displacement capability isemployed in the performance of the method in order that strands areappropriately displaced to affect the amplification process. The term“strand displacement” refers to the ability of a polymerase to displacedownstream DNA encountered during synthesis. A range of polymerases withstrand displacement capability that operate at different temperatureshave been characterised and are commercially available. For example,Phi29 polymerase has a very strong ability to strand displace.Polymerases from Bacillus species, such as Bst DNA Polymerase LargeFragment, typically exhibit high strand displacing activity and arewell-suited to use in the performance of the method. E. coli Klenowfragment (exo −) is another widely used strand displacement polymerase.Strand displacement polymerases may be readily engineered, such asKlenTaq such as by cloning of only the relevant active polymerase domainof an endogenous enzyme and knock-out of any exonuclease activity. Forthe performance of the method wherein the single stranded target nucleicacid is RNA, RNA dependent DNA synthesis (reverse transcriptase)activity is also required, which activity may be performed by the stranddisplacement polymerase and/or by a separate additional reversetranscriptase enzyme in step a), e.g. M-MuLV or AMV.

Polymerase(s) are typically employed in the relevant steps of the methodin an appropriate amount which is optimised dependent on the enzyme,concentration of reagents and desired temperature of the reaction. Forexample, of 0.1-100 Units of a Bacillus polymerase may be used, whereone unit is defined as the amount of enzyme that will incorporate 25nmol of dNTP into acid insoluble material in 30 minutes at 65° C.However, the amount depends on a number of factors such as the activityof the polymerase, its concentration and form, the anticipatedconcentration of the target nucleic acid, the volume of the reaction,the number and concentration of the oligonucleotide primers and thereaction temperature, and should not be considered limiting in any way.

Those skilled in the art will know that polymerases require dNTPmonomers to have polymerase activity and also that they require anappropriate buffer, with components such as buffer salts, divalent ionsand stabilising agents. In addition, one or more modified dNTP is usedin the method in order to block the cleavage of the reversecomplementary strand of the primers following incorporation by thestrand displacement polymerase. Typically when a single modified dNTP isused, the dNTPs used in the method shall omit the corresponding base.For example, in an embodiment in which the modified dNTP is alpha thioldATP, the dNTPs shall comprise only dTTP, dCTP and dGTP and shall notinclude dATP. Removing the corresponding natural dNTP base ensures thatthe all of the required bottom strand cleavage sites within the reversecomplementary sequence of the primers are blocked because only themodified base is available for incorporation by the polymerase, howevercomplete or partial removal of the corresponding natural dNTP base isnot essential. dNTPs may typically be used in the method at similarconcentrations to those employed in other polymerase methods, such asconcentrations ranging from 10 micromolar to 1 millimolar, although theconcentration of dNTP for the method may be optimised for any givenenzyme and reagents, in order to maximise activity and minimise abinifio synthesis to avoid background signal generation. Given thatcertain polymerases can exhibit a lower rate of incorporation with oneor more modified dNTP base the one or more modified base may be used inthe method at a higher relative concentration that the unmodified dNTPs,such as at a five-fold higher concentration, although this should beconsidered non-limiting.

The use of one or more modified dNTP is an integral feature of thepresent invention which offers an important advantage in addition toproviding for the restriction enzymes to cleave only one strand of theirrestriction sites. For example, certain modified dNTPs, such as alphathiol dNTPs, lead to a reduction in the melting temperature (Tm) of theDNA into which they are incorporated which means the oligonucleotideprimers and probes used in the method have a greater affinity forhybridisation to species within the amplification product than anycompeting modified dNTP complementary strands produced during theamplification. This key feature enhances the amplification rate because,for example, when one of the displaced strands hybridises to its reversecomplement to produce an “unproductive” end-point species, it morereadily dissociates than the “productive” hybridisation of saiddisplaced strand to a further primer due to the presence of one or moremodified bases leading to a reduction in the Tm of hybridisation. It hasbeen reported that phosphorothioate internucleotide linkages can reducethe Tm, the temperature at which exactly one half the single strands ofa duplex are hybridised, by 1-3° C. per addition, a substantial changein the physicochemical properties. We have also observed an enhancedrate of strand displacement when phosphorothioate nucleotide linkagesare present in a DNA sequence. Furthermore, the oligonucleotide probesused in the method, whether contacted with the sample simultaneously tothe performance of step a) or subsequently, possess a higher affinityfor those species within the amplification product than any competingmodified species and can thus preferentially hybridise or even displacehybridised strands to facilitate production of the detector species. Thereduced Tm and enhanced displacement of amplification product species asa result of the modified internucleotide linkages they contain serve tofundamentally enhance the rate of the method and reduce the temperaturerequired for rapid amplification to occur.

In addition to the rate enhancement resulting from the use of one ormore modified nucleotide, the specificity of hybridisation of theoligonucleotide primers and probes of the method is also enhanced. Giventhat typically all of the bases of one particular nucleotide aresubstituted within amplification product, the hybridisation sites of theprimers and probes typically contain modified bases and the reduced Tmresulting from phosphorothioate internucleotide linkages, for example,means that sequence mismatches from non-specific hybridisation are lesslikely to be tolerated.

Thus the integral feature of the method of the invention for one or moremodified dNTP leads to fundamental benefits that enhance both thesensitivity and specificity of amplification and are in stark contrastto known methods without such a requirement for modified nucleotides,such as NEAR (WO2009/012246), including NEAR variants with softwareoptimised primers (WO2014/164479) or a warm start or controlledreduction in temperature (WO2018/002649).

A number of different modified dNTPs, such as modified dNTPs that confernuclease resistance following their incorporation by a polymerase, existand can be employed in the method to accomplish resistance to cleavageby the restriction enzyme and, in embodiments, other features to enhancethe performance of the method for a given application. In addition toalpha thiol dNTPs which provide for nuclease resistance and a reductionin Tm, modified dNTPs that are reported to have potential for polymeraseincorporation and to confer nuclease resistance, include equivalentnucleotide derivatives, such as Borano derivatives, 2′-O-Methyl (2′OMe)modified bases and 2′-Fluoro bases. Other modified dNTPs or equivalentcompounds that may be incorporated by polymerases and used inembodiments of the method to enhance particular properties of themethod, include those that decrease binding affinity, e.g.Inosine-5′-Triphosphate or 2′-Deoxyzebularine-5′-Triphosphate, thosethat increase binding specificity, e.g.5-Methyl-2′-deoxycytidine-5′-Triphosphate or5-[(3-Indolyl)propionamide-N-allyl]-2′-deoxyuridine-5′-Triphosphate, andthose that enhance the synthesis of GC rich regions, e.g. 7-deaza-dGTP.Certain modifications can increase Tm providing further potential forcontrol of the hybridisation events in embodiments of the method.

Steps a), b) and c) may be performed over a wide range of temperatures.The optimal temperature for each step is determined by the temperatureoptimum of the relevant polymerase and restriction enzymes and themelting temperature of the hybridising regions of the oligonucleotideprimers. Notably the method does not use temperature cycling in step a).Furthermore, the amplification step a) does not require any controlledoscillation of temperature, nor any hot or warm start, pre-heating or acontrolled temperature decrease. The method allows the steps to beperformed over a wide temperature range, e.g. 15° C. to 60° C., such as20 to 60° C., or 15 to 45° C. According to an embodiment, step a) isperformed at a temperature of not more than 50° C., or about 50° C.Given the wide range of restriction enzymes that are not nicking enzymesavailable for use in the method, it is possible to select restrictionenzymes with a rapid rate at relatively low temperatures compared toalternative methods using nicking enzymes. The use of one or moremodified nucleotides also reduces the temperature of amplificationrequired. In addition to having the potential for a lower optimaltemperature profile compared to known methods, the method of theinvention can be performed over an unusually broad range oftemperatures. Such features are highly attractive for use of the methodin a low-cost diagnostic device, where controlled heating imposescomplex physical constraints that increase the cost-of-goods of such adevice to a point where a single-use or instrument-free device is notcommercially viable. A number of assays have been developed using themethod that can perform rapid detection of target nucleic acid atambient temperature or at around 37° C., for example. As such, in afurther embodiment step a) is performed at a temperature of not morethan 45° C., or about 45° C. It may be preferable to initiate the methodat a temperature lower than the targeted temperature in order tosimplify the user steps and decrease the overall time to result. As suchin a further embodiment of the method, the temperature of step a) isincreased during the amplification. For example, the temperature of themethod may start at ambient temperature, such as 20° C., and increaseover a period, such as two minutes, to the final temperature, such asapproximately 45° C. or 50° C. In an embodiment the temperature isincreased during the performance of step a), such as an increase from anambient starting temperature, e.g. in the range of 15-30° C., up to atemperature in the range of 40-50° C.

The low temperature potential and versatility of the method of theinvention means that, in contrast to known methods, it is compatiblewith the conditions required for a range of other assays, such asimmunoassays or enzymatic assays for the detection of other biomarkers,such as proteins or small molecules. Therefore the method can be used,for example, for the simultaneous detection of both nucleic acids andproteins or small molecules of interest within a sample. The componentsrequired for performance of the method, including restriction enzymesthat are not nicking enzymes, strand displacement DNA polymerase,oligonucleotide primers, oligonucleotide probes, dNTPs and one or moremodified dNTP, may be lyophilised or freeze-dried for stable storage andthe reaction may then be triggered by rehydration, such as upon additionof the sample. Such lyophilisation or freeze-drying for stable storagetypically requires addition of one or more excipients, such astrehalose, prior to drying the components. A very wide range of suchexcipients and stabilisers for lyophilisation or freeze-drying are knownand available for testing in order to identify a suitable compositionfor the components required for the performance of the method.

It will be apparent to one skilled in the art that the method of theinvention, being a polymerase-based amplification method, may beenhanced by the addition of one or more additive that has been shown toenhance PCR or other polymerase based amplification methods. Suchadditives include but are not limited to tetrahydrothiophene 1-oxide,L-lysine free base, L-arginine, glycine, histidine, 5-aminovaleric acid,1,5-diamino-2-methylpentane, N,N′-diisopropylethylenediamine,tetramethylenediamine (TEMED), tetramethylammonium chloride,tetramethylammonium oxylate, methyl sulfone acetamide,hexadecyltrimethylammonium bromide, betaine aldehyde,tetraethylammoniumchloride, (3-carboxypropyl)trimethylammoniumchloride,tetrabutylammoniumchloride, tetrapropylammoniumchloride, formamide,dimethylformamide (DMF), N-methylformamide, N-methylacetamide,N,N-dimethylacetamide, L-threonine, N,N-dimethylethylenediamine,2-pyrrolidone, HEP (N-hydroxyethylpyrrolidone), NMP(N-methylpyrrolidone) and 1-methyl, 1-cyclohexyl-2-pyrrolidone(pyrrolidinones), 6-valerolactam, N-methylsuccinimide,1-formylpyrrolidine, 4-formylmorpholine, DMSO, sulfolane, trehalose,glycerol, Tween-20, DMSO, betaine and BSA.

Our investigations have revealed that the present method is effectiveover a wide range of target nucleic acid levels including detection downto very low, even single, copy numbers. The oligonucleotide primers aretypically provided in vast excess over target nucleic acid. Typicallythe concentration of each primer is in the range 10 to 200 nM althoughthat should be considered non-limiting. A higher primer concentrationcan enhance the efficiency of hybridisation and therefore increase therate of the reaction. However, non-specific background effects, such asprimer dimers, can also be observed at high concentration and thereforethe concentration of the first and second oligonucleotide primers formspart of the optimisation process for any given assay employing themethod. In an embodiment the first and the second oligonucleotideprimers are provided at the same concentration. In an alternativeembodiment one of the first and second oligonucleotide primers isprovided in excess of the other. The rate of reaction may be reduced inembodiments wherein one of the primers is provided in excess of theother due to the natural symmetry of the cyclical amplification process,however in certain circumstances it can be used to reduce non-specificbackground signal in the method and/or to enhance the ability of thefirst and second oligonucleotide probes to hybridise to produce thedetector species. It is desirable that both primers are present at suchas level as to not become limiting before sufficient detector specieshas been produced for detection with the selected means of detection.

There are a number of considerations for the design of theoligonucleotide primers for performance of the method. Each of the firstand second oligonucleotide primers must comprise in the 5′ to 3′direction one strand of a restriction enzyme recognition sequence andcleavage site and a hybridising region, wherein said hybridising regionis capable of hybridising to a first hybridisation region in the targetnucleic acid in the case of the first primer and to the reversecomplement of a second hybridisation sequence upstream of the firsthybridisation sequence in the target nucleic acid in the case of thesecond primer. Thus a pair of primers is designed to amplify a region ofthe target nucleic acid. The restriction enzyme recognition sequence ofthe primers is not typically present within the target nucleic acidsequence and thus forms an overhang during the initial hybridisationevents before being introduced to the amplicon (see FIG. 1). In theevent that an asymmetric restriction enzyme is used the cleavage site istypically downstream of the recognition sequence and may therefore,optionally, be present within the hybridising sequence of the primer.

The oligonucleotide primers are designed such that following theircleavage in the method, the sequence 5′ of the cleavage site forms anupstream primer with sufficient melting temperature (Tm) to remainhybridised to its reverse complementary strand under the desiredreaction conditions and to displace the strand downstream of thecleavage site following extension of the 3′ hydroxyl group by the stranddisplacement DNA polymerase. Thus an additional “stabilising” region maybe included at the 5′ end of the oligonucleotide primers, the optimumlength of which is determined by the position of the cleavage siterelative to the recognition sequence for the relevant restriction enzymeand other factors such as the temperature to be employed for theamplification in step a). Thus in an embodiment the first and/or secondoligonucleotide primers comprise a stabilising sequence upstream of therestriction enzyme recognition sequence and cleavage site, such as atthe 5′ end, and e.g. of 5 or 6 bases in length.

During primer design it is necessary to define the sequence and lengthof each hybridising region in order to permit optimal sequence specifichybridisation and strand displacement to ensure specific and sensitiveamplification in the method. The positioning of the primers within thetarget nucleic acid to be detected, e.g. within the genome of a viral orbacterial pathogen, may be varied to define the sequence of thehybridising region of the primers and thus to select primers with theoptimal sensitivity and specificity for amplification and compatibilitywith the oligonucleotide probes. Different primer pairs can therefore bescreened to identify the optimal sequence and positioning forperformance of the method. Typically the length of the hybridisingregion of the primers is designed such that its theoretical Tm permitsefficient hybridisation at the desired reaction temperature but is alsoreadily displaced following cleavage. During primer design, thetheoretical Tm of the hybridising sequence and the sequence of thedisplaced strands are considered in the context of the likelytemperature of the reaction and the restriction enzyme selected, whichis balanced with the theoretical improvement to sequence-derivedspecificity of binding that can result as sequence length is increased.Our various investigations have indicated considerable versatility inthe design of the primers to be used effectively in the method. In anembodiment the hybridising region of the first and/or secondoligonucleotide primers is between 6 and 30, e.g. 9 and 16, bases inlength. In further embodiments modifications, such as non-natural basesand alternative internucleotide linkages or abasic sites may be employedin the hybridising regions of the primers to refine their properties andthe functioning of the method for a particular application. For examplea modification that enhances Tm, such as PNA, LNA or G-clamp may permita shorter and more specific primer hybridisation region which enables ashorter amplicon and thus enhances the rate of amplification.

Our various investigations have revealed that the rate of the method andits sensitivity may be enhanced by having a short amplicon and thus incertain embodiments it can be preferable to shorten both the overalllength of the primers, including their hybridising sequence, and toposition the primers with only a short gap, such as 10 or 15 nucleotidebases or less, between the first and second hybridisation sequences inthe target nucleic acid. In an embodiment the first and secondhybridisation sequences in the target nucleic acid are separated by 0 to15 or 0 to 6 bases, in certain embodiments they are separated by 3 to 15or 3 to 6 bases, e.g. 5, 7 or 11 bases. In a further embodiment thehybridisation sequences are overlapping, such as by 1 to 2 bases.

There are a number of considerations to the design of the sequence ofthe oligonucleotide probes for use in the method. Firstly, the region inthe first oligonucleotide probe hybridising to the first single strandeddetection sequence and the region in the second oligonucleotide probehybridising to the second single stranded detection sequence aretypically designed such that they are non-overlapping or have minimaloverlap, to permit both oligonucleotide probes to bind at the same timeto the at least one species within the amplification product. They arealso typically designed to hybridise mainly to sequence that fallsbetween the position of the cleavage site in one strand of theamplification product species and the position opposite the cleavagesite on the reverse complementary strand thereto in order to ensure theone or more species within the amplification product are efficientlytargeted and that both oligonucleotide probes bind to the same strand.For any given pair of primers, either strand may be selected fortargeting by the oligonucleotide probes. Given that the oligonucleotideprobes are not typically extended by a polymerase in the method, thehybridising sequences are designed based upon the relevant sequence ofthe species within the amplification product, which determines their Tm,% GC and the experimental performance data obtained. In an embodiment,the hybridising sequence of the first and second oligonucleotide probesis 9 to 20 nucleotide bases long. In an embodiment wherein the first andsecond hybridisation sequences in the target nucleic acid are separatedby 0 bases, the sequence of the hybridising regions of one of theoligonucleotide probes may correspond to one of the oligonucleotideprimers and the hybridising region of the other oligonucleotide probewould correspond to the reverse complement of the other oligonucleotideprimer. However, the length of the hybridising sequences may betruncated in order to optimise the properties of the oligonucleotideprobes for the desired embodiment of the method and avoid any inhibitoryeffects in the event that all or part of step b) is performedsimultaneously to step a). In the event that the first or secondoligonucleotide probe encompasses a recognition sequence and cleavagesite for either the first or second restriction enzyme and saidoligonucleotide probe is contacted with the sample simultaneously to theperformance of step a), the cleavage site within said probe is typicallyblocked, for example by the inclusion of a modified internucleotidelinkage, e.g. a phosphorothioate linkage, during the chemical synthesisof the probe or introduction of a mismatch to remove said recognitionsequence. Other than the hybridising regions, there is considerableversatility to the sequence of the oligonucleotide probes and to anymodified nucleotide bases, nucleotide linkages or other modificationsthat they may comprise. Modified bases that may be chemically insertedinto oligonucleotides to alter their properties and may be employed inembodiments of the methods, such as 2-Amino-dA, 5-Methyl-dC, Super T®,2-Fluoro bases and G clamp provide for an increase in Tm, whilst otherssuch as Iso-dC and Iso-G, can enhance specificity of binding withoutincreasing Tm. Other modifications such as inosine or abasic sites maydecrease the specificity of binding. Modifications known to confernuclease resistance include inverted dT and ddT and C3 spacers.Modifications can increase or decrease Tm and provide potential forcontrol of the hybridisation events in embodiments of the method. Use ofmodified bases within the hybridising regions of the oligonucleotideprobes provides an opportunity to improve the performance of theoligonucleotide probes such as by enhancing their binding affinitywithout increasing the length of the hybridising region. In anembodiment modified bases within one or both oligonucleotide probespermit them to hybridise more effectively than, and thus out-compete,any species within the amplification product with complementarity to therelevant single stranded detection sequence.

In embodiments wherein one of the first and second oligonucleotideprobes is blocked at the 3′ end from extension and is not capable ofbeing cleaved and is contacted with the sample simultaneously to theperformance of step a), typically said one oligonucleotide probe willcomprise an additional 5′ region, which provides the opportunity for thestabilisation of the pre-detector species as described (see FIG. 2). Inan embodiment said one oligonucleotide probe comprises the exactsequence of one of the oligonucleotide primers, but contains amodification at the 3′ end to block its extension by the stranddisplacement DNA polymerase and a single phosphorothioateinternucleotide linkage to block the restriction enzyme cleavage site.Such an embodiment simplifies assay design and ensures that noadditional sequence motifs are introduced which may lead to non-specificbackground amplification.

The first and second oligonucleotide probes that produce the detectorspecies are preferably provided at a level wherein the number of copiesof detector species produced is sufficiently above the limit ofdetection of the means employed for said detector species to be readilydetected. Furthermore the efficiency of hybridisation by the firstand/or second oligonucleotide probe(s) are influenced by theirconcentration. Typically the concentration of an oligonucleotide probecontacted with the sample simultaneously to the performance of step a)may be similar to the concentration of the oligonucleotide primers, e.g.10 to 200 nM, although that should be considered non-limiting. In anembodiment the concentration of one or both oligonucleotide probes isprovided in excess of the concentration of one or both oligonucleotideprimers, whist in another embodiment the concentration of one or botholigonucleotide probes is provided at a lower concentration than one orboth oligonucleotide primers. In the event one or both oligonucleotideprobes is contacted to the sample subsequent to the performance of theamplification step a), a higher concentration may be permitted asnecessary to accomplish the most efficient hybridisation, without anyconsideration of inhibition to the amplification step a) that mayresult.

Hybridisation sequences are a key feature of both the oligonucleotideprimers and oligonucleotide probes for performance of the method.Hybridisation refers to sequence specific hybridisation which is theability of an oligonucleotide primer or probe to bind to a targetnucleic acid or species within the amplification product by virtue ofthe hydrogen bond base pairing between complementary bases in thesequence of each nucleic acid. Typical base pairings are Adenine-Thymine(A-T), or Adenine-Uracil in the case of RNA or RNA/DNA hybrid duplexes,and Cytosine-Guanine (C-G), although a range of natural and non-naturalanalogues of nucleic acid bases are also known with particular bindingpreferences. Furthermore, in the present invention, the complementarityregion of an oligonucleotide probe or primer does not necessarily needto comprise wholly natural nucleic acid bases in a sequence withcomplete and exact complementarity to its hybridisation sequence in thetarget nucleic acid or species within the amplification product; ratherfor the performance of the method the oligonucleotide probes/primersonly need to be capable of sequence specific hybridisation to theirtarget hybridisation sequence sufficiently to form the double strandedsequence necessary for the correct functioning of the method, includingthe cleavage by the restriction enzymes and extension by the stranddisplacement DNA polymerase. Therefore such hybridisation may bepossible without exact complementarity, and with non-natural bases orabasic sites. In an embodiment, the hybridising regions of anoligonucleotide primer or oligonucleotide probe used in the method mayconsist of complete complementarity to the sequence of the relevantregion of the target nucleic acid or species within the amplificationproduct, or its reverse complementary sequence, as appropriate. In otherembodiments there are one or more non-complementing base pairs. In somecircumstances it may be advantageous to use a mixture of oligonucleotideprimers and/or probes in the method. Thus, by way of example, in thecase of a target nucleic acid comprising a single nucleotidepolymorphism (SNP) site having two polymorphic positions, a 1:1 mixtureof oligonucleotide primers and oligonucleotide probes differing in thatposition (each component having complementarity to the respective baseof the SNP) may be employed. During manufacture of oligonucleotides itis routine practice to randomise one or more bases during the synthesisprocess.

One skilled in the art will understand that amplification processesinvolving polymerases can suffer from non-specific backgroundamplification such as that resulting from ab initio synthesis and/orprimer-primer binding. Whilst the method of the invention typicallyexhibits more rapid amplification when the length of amplicon isdesigned to be as short as possible, e.g. by minimising the hybridisingsequences of the primers, the gap between the first and secondhybridisation sequences in the target nucleic acid and the length of anystabilising region, to the extent possible whilst still retainingfunction at the given reaction temperature. With shorter ampliconsnon-specific background may be exacerbated due to the fact that allnecessary sequence to produce the amplification product species isprovided by the oligonucleotide primers. In the event an amplicon isproduced in a non-target specific manner comprising both the firstoligonucleotide primer and the second oligonucleotide primer “connected”via an ab initio synthesised DNA or primer-primer binding, a falsepositive result could occur in the method. The use of twooligonucleotide probes in the present method allows for a variety ofembodiments of the method encompassing additional features to minimiseany possibility of non-target specific background signal. Suchembodiments made possible by the use of two oligonucleotide probespresent a substantial advantage over known methods in this regard.

One approach is to separate the first and second hybridisation sequencesin the target nucleic acid to provide a target-based sequencespecificity check using the oligonucleotide probes of the method. Thusin an embodiment, the first and second hybridisation sequences in thetarget nucleic acid are separated by 3 to 15 or by 3 to 6 bases, e.g. 5,7 or 11 bases. This gap between the primers presents the optimal sizegap to provide for an additional specificity check on species within theamplification product whilst still maintaining the enhanced rate of ashort amplicon. Thus in an embodiment, in step b) either the first orsecond single stranded detection sequence in the at least one specieswithin the amplification product includes at least 3 bases of thesequence corresponding to said 3 to 15 or 3 to 6 bases. For example, wehave demonstrated the potential to distinguish a specifictarget-dependent amplification product from non-target specificbackground amplification products, as shown in Example 4 (FIG. 8).

In an alternative approach the concentration of the first and/or secondoligonucleotide primers is decreased to reduce the probability ofbackground resulting from ab initio amplification and from primer-primerbinding. In order to ensure the rate of the amplification is maintained,additional oligonucleotide primers that are blocked at the 3′ end fromextension by the strand displacement DNA polymerase may be used. In thisembodiment, whilst the unblocked first and second oligonucleotideprimers are available at sufficient concentration for the initialhybridisation and extension events to produce the amplicon from thetarget nucleic acid, subsequent amplification proceeds with the blockedprimers, which are preferably provided at higher concentration, whereincleavage of the blocked primers occurs prior to their extension andstrand displacement in order to remove the 3′ blocking modification andallow the amplification process to proceed without detriment (see FIG.4). Thus in an embodiment, the sample additionally is contacted in stepa) with: (A) a third oligonucleotide primer which third primer comprisesin the 5′ to 3′ direction one strand of the recognition sequence andcleavage site for the first restriction enzyme and a region that iscapable of hybridising to the first hybridisation sequence in the targetnucleic acid and wherein said third primer is blocked at the 3′ end fromextension by the DNA polymerase; and/or (B) a fourth oligonucleotideprimer which fourth primer comprises in the 5′ to 3′ direction onestrand of the recognition sequence and cleavage site for the secondrestriction enzyme and a region that is capable of hybridising to thereverse complement of the second hybridisation sequence in the targetnucleic acid and wherein said fourth primer is blocked at the 3′ endfrom extension by the DNA polymerase. In a further embodiment, whenpresent the third oligonucleotide primer is provided in excess of thefirst oligonucleotide primer and when present the fourth oligonucleotideprimer is provided in excess of the second oligonucleotide primer. Byreducing the concentration of the first and second oligonucleotideprimers substantially, offset by the presence of the third and fourtholigonucleotide primers, the maximum potential benefit in terms ofremoval of non-target dependent background amplification is obtained.Other than the presence of the 3′ modification to block polymeraseextension which may readily be achieved through, for example, use of a3′ phosphate or C-3 modification during oligonucleotide primersynthesis, the same design parameters as employed for the first andsecond primers apply to the third and fourth primers.

Embodiments of the method of the invention that provide for enhancedspecificity and removal of background amplification as described above,provide improved rigour of sequence verification, which enables lowtemperature reactions to be performed without loss of specificity and/orenables increased multiplexing, where multiple reactions are performedfor the simultaneous detection of multiple targets. The benefits of thisrigorous specificity also mean that the method can tolerate a broadtemperature range and suboptimal conditions (e.g. reagentconcentrations) without loss of specificity. For example, we haveperformed the method with a 20% increase or decrease in theconcentration of all components and we have performed the method with asubstantial period at ambient temperature following performance of theamplification in step a) in each case without any loss of specificityobserved. Therefore such embodiments represent important advantages ofthe method of the invention over known methods and mean that it isideally suited to exploitation in a low-cost and/or single-usediagnostic device.

Detection of the detector species in step c) can be accomplished by anytechnique which differentially detects the presence of the detectorspecies from the other reagents and components present in the sample.Alternatively the presence or level of the detector species can beinferred from the depletion of one or more reaction components such asthe first or second oligonucleotide probe. From a wide range ofphysicochemical techniques available for use in the detection of thedetector species, those capable of generating a sensitive signal thatonly exists following hybridisation of the first oligonucleotide probeand second oligonucleotide probe to the relevant species in theamplification product are prioritised for use in the method. It will beapparent to a skilled person that a range of colorimetric orfluorometric dyes exist that may be readily attached to the firstoligonucleotide probe and form the basis of its detection, eithervisually or using instrumentation, such as absorbance or fluorescencespectroscopy.

Thus in an embodiment, the moiety that permits the detection of thefirst oligonucleotide probe, is a colorimetric or fluorometric dye or amoiety that is capable of attachment to a colorimetric or fluorometricdye such as biotin.

Embodiments of the method employing colorimetric dyes have the advantageof not requiring an instrument to perform fluorescence excitation anddetection and potentially of allowing the presence of the target nucleicacid to be determined by eye. Colorimetric detection can be achieved bydirectly attaching a colorimetric dye or moiety capable of attachment toa colorimetric dye to the first oligonucleotide probe prior to its usein the method, or alternatively specifically attaching or binding thedye or moiety to the probe fragment following cleavage. For example, thefirst oligonucleotide probe may contain a biotin moiety that permits itsbinding to a streptavidin conjugated colorimetric dye for its subsequentdetection. One such example of a colorimetric dye that may be used indetection is gold nanoparticles Similar methods can be employed with avariety of other intrinsically colorimetric moieties, of which a verylarge number are known, such as carbon nanoparticles, silvernanoparticles, iron oxide nanoparticles, polystyrene beads, quantum dotsetc. A high extinction coefficient dye also provides potential forsensitive real-time quantification in the method.

A number of considerations are taken into account when choosing anappropriate dye for a given application. For example, in embodimentswhere it is intended to perform visible colorimetric detection insolution, it would generally be advantageous to choose larger sizeparticles and/or those with a higher extinction coefficient for ease ofdetection, whereas embodiments incorporating a lateral flow membraneintended for visible detection, might benefit from the ability ofsmaller sized particles to more rapid diffuse along a membrane. Whilevarious sizes and shapes of gold nanoparticles are available, a numberof other colorimetric moieties of interest are also available whichinclude polystyrene or latex based microspheres/nanoparticles. Particlesof this nature are also available in a number of colours, which can beuseful in order to tag and differentially detect different detectorspecies during the performance of the method, or “multiplex” thecolorimetric signal produced in a detection reaction.

Fluorometric detection can be achieved through the use of any dye thatunder appropriate excitation stimulus, emits a fluorescent signalleading to subsequent detection of the detector species. For example,dyes for direct fluorescence detection include, without limitation:quantum dots, ALEXA dyes, fluorescein, ATTO dyes, rhodamine and texasred. In embodiments of the method that employ a fluorescent dye moietyattached to an oligonucleotide probe, it is also possible to performdetection based on fluorescence resonance energy transfer (FRET), suchas employed in Taqman quantitative PCR or Molecular Beacon basedstrategies for nucleic acid detection, whereby the signal would increaseor decrease following attachment of the dye to the detector species.Generally, when a fluorometric approach is used a number of differentdetector devices can be used to record the generation of fluorescentsignal, such as for example CCD cameras, fluorescence scanners,fluorescence based microplate readers or fluorescence microscopes.

In a further embodiment the moiety that permits the detection of thefirst oligonucleotide probe is an enzyme that yields a detectablesignal, such as a colorimetric or fluorometric signal, following contactwith a substrate. It will be apparent to a skilled person that a numberof enzyme substrate systems are available and routinely used in thefield of diagnostics, such as in ELISA and Immunohistochemistrydetection. Horseradish peroxidase (HRP) is one example Utilising anenzyme attached to the first oligonucleotide probe for detection of thedetector species in step c), offers a number of potential advantages,such as enhanced sensitivity of detection and increased control ofsignal development through a separate step involving addition ofsubstrate. Other suitable colorimetric enzymes might include: glycosylhydrolases, peptidases or amylases, esterases (e.g. carboxyesterase),glycosidases (e.g. galactosidase), and phosphatases (e.g. alkalinephosphatase). This list should not be considered in any way limiting.

In another approach, the presence of the detector species in step c) isdetected electrically, such as by a change in impedence or a change inconductimetric, amperometric, voltammetric or potentiometric signal, inthe presence of the detector species. Thus in an embodiment the detectorspecies is detected by a change in electrical signal. The electricalsignal change may be facilitated by the moiety that permits thedetection of the first oligonucleotide probe, such as a chemical groupthat leads to an enhanced change in electrical signal. Since electricalsignal detection can be so sensitive said detection moiety may be simplyan oligonucleotide sequence, although in certain embodiments signal isenhanced by the presence of chemical groups known to enhance electricalsignals, such as metals e.g. gold and carbon.

Whilst in an embodiment the electrical signal change resulting fromaccumulation of the detector species may be detected in an aqueousreaction during amplification, in other embodiments the electricalsignal detection is facilitated by the localisation of the detectorspecies to a particular site for its detection, such as the surface ofan electrochemical probe, wherein said localisation is mediated by thesecond oligonucleotide probe.

Other techniques that are routinely employed for the detection ofnucleic acids such as the detector species and may also be employed fordetection in the method include: mass spectrometry (such as MALDI orLC-TOF), luminescence spectroscopy or spectrometry, fluorescencespectroscopy or spectrometry, liquid chromatography and fluorescencepolarization.

In an embodiment, step c) produces a colorimetric or electrochemicalsignal using carbon or gold, preferably carbon.

In an embodiment the detector species is detected by nucleic acidlateral flow. Nucleic acid lateral flow, wherein nucleic acids areseparated from other reaction components by their diffusion through amembrane, typically made of nitrocellulose, is a rapid and low-costmethod of detection capable of coupling with a range of signalread-outs, including colorimetric, fluorometric and electrical signals.Nucleic acid lateral flow is well suited for use in the detection of thedetector species in the method and offers a number of advantages. In anembodiment the nucleic acid lateral flow detection is performed whereinthe first oligonucleotide probe within the detector species is used toattach a colorimetric or fluorometric dye and the second oligonucleotideprobe within the detector species is used to localise said dye to adefined location on the lateral flow strip. In this way, rapid detectioncan be performed with results visualised by eye or by a readerinstrument. Nucleic acid lateral flow may employ an antigen as thedetector moiety in the second oligonucleotide probe with the associatedantibody immobilised on the lateral flow strip. Alternatively in thepresent method sequence specific detection via hybridisation of thepre-detector species or detector species onto the lateral flow strip maybe readily performed providing for a simple, low cost alternative toantibody based assays with improved multiplexing potential. Knownmethods, such as SDA, that do not utilise the two oligonucleotide probesof the present method, typically generate double stranded DNA productswhich are not available for detection based upon sequence specifichybridisation. In contrast in the present method, the detector speciesis particularly amenable to multiplex detection, by virtue of the use oflocation specific hybridisation based detection. Carbon or goldnanoparticles may be readily employed in nucleic acid lateral flow.Localisation of the detector species causes local concentration ofcarbon or gold, causing appearance of a black or red colour,respectively. In an embodiment the first oligonucleotide probe containsa moiety, such as a biotin, that permits its binding to a colorimetricdye prior to localisation on the strip by sequence specifichybridisation.

The spatial positioning of the detector species is closely associatedwith the technique employed for detection of the detector species, as itpermits, for example, the hybridisation based binding of the detectorspecies at a particular location. In addition to facilitating rapid andspecific detection, such physical attachment can enhance the use of themethod in the multiplex detection of multiple different target nucleicacids. In an embodiment the second oligonucleotide probe is attached ona nucleic acid lateral flow strip or on the surface of anelectrochemical probe, a 96-well plate, beads or an array surface. Thusthe at least one species within the amplification product becomeslocalised to the physical location of the second oligonucleotide probewhich is readily detected following the formation of the detectorspecies at such location. Alternatively, it can be advantageous to use asingle stranded oligonucleotide as the moiety attached to the secondoligonucleotide probe that permits its attachment to a solid material.In this way the sequence of the solid phase attached oligonucleotide canbe defined independently to the target nucleic acid sequence to enhancethe efficiency of binding. Thus, in an embodiment the moiety thatpermits the attachment of the second oligonucleotide probe to a solidmaterial is a single stranded oligonucleotide. Said single strandedoligonucleotide can be designed to have improved affinity and efficiencyof hybridisation to enhance performance of the method. For example, incertain embodiments of the method rather than attaching the secondoligonucleotide probe to the lateral flow strip directly, a separateoligonucleotide with a sequence optimised for on-strip hybridisation isemployed that is capable of efficient hybridisation to the singlestranded oligonucleotide moiety present within the secondoligonucleotide probe.

In various investigations we have significantly enhanced performance ofthe method by nucleic acid lateral flow using a single strandedoligonucleotide as the attachment moiety of the second oligonucleotideprobe, which provides for the on-strip hybridisation sequence to beenhanced. For example, a G-C rich sequence may be employed for theon-strip hybridisation, or a longer sequence with higher Tm may beemployed, that supplements the length of the second oligonucleotideprobe. Alternative, said single stranded oligonucleotide moiety maycomprise one or more modified base or internucleotide linkage to enhanceits affinity, such as a PNA, LNA or G-clamp We have observed that when arepeating sequence motif is employed in the single strandedoligonucleotide moiety, a surprising enhancement of the hybridisationefficiency is observed which is not predicted by its predicted Tm. Thusin an embodiment the sequence of the single stranded oligonucleotidemoiety comprises three or more repeat copies of a 2 to 4 base DNAsequence motif. For example, in various investigations employing such asequence motif we have observed a substantial enhancement in thesensitivity of detection by nucleic acid lateral flow, frequently with asignal enhancement of 100-fold or more.

Thus in an embodiment wherein the presence of the detector species isdetected by nucleic acid lateral flow, the nucleic acid lateral flowutilises one or more nucleic acids that is capable of sequence specifichybridisation to the moiety that permits the attachment of the secondoligonucleotide probe to a solid material.

A further advantage is conferred by de-coupling the target nucleic acidsequence from the solid material for attachment or from the means ofdetection, this may be permitted by the use of the single strandedoligonucleotide as the detection moiety within the first oligonucleotideprobe and/or the attachment moiety with the second oligonucleotideprobe. In this way the relevant solid material for attachment, or devicecontaining said solid material, such as the nucleic acid lateral flowstrip, and/or the means of detection, can be optimised and definedwithout regard to the sequence of the target nucleic acid to bedetected. Such a “universal” detection apparatus can be used fromapplication to application and target to target without needing to bealtered. For example a nucleic acid lateral flow strip with printedlines corresponding to a compatible set of oligonucleotide sequenceswhich have the ability for efficient on-strip hybridisation and nounintended cross-talk can be defined, optimised and efficientlymanufactured independently of the development of the oligonucleotideprimers and probes of the method for detection of multiple targetnucleic acid sequences.

In a number of embodiments detection may be performed in a quantitativemanner Thus, the level of the single stranded target nucleic acid in thesample may be quantified in step c). Quantification may be accomplishede.g. by measuring the detector species colorimetrically,fluorometrically or electrically, during the time course of the reactionat multiple time-points rather than at a single end-point. Alternativestrategies for quantification include sequential dilution of the sample,analogous to droplet digital PCR. In a further embodiment the level ofthe single stranded target nucleic acid in the sample may be determinedsemi-quantitatively. For example, where the intensity of a colorimetricsignal on a nucleic acid lateral flow strip would correspond to theapproximate level of the single stranded target nucleic acid in thesample. Alternatively an inhibitor may be used whereby the number ofcopies of the single stranded nucleic acid target must exceed a certaindefined number of copies in order to overcome the inhibitor and producea detectable number of copies of the detector species.

In the method of the invention the second oligonucleotide probe isattached to a solid material or to a moiety that permits its attachmentto a solid material. Optionally, in embodiments, one or more of theother oligonucleotide primers and probes may also be attached to a solidmaterial or to a moiety that permits their attachment to a solidmaterial. It will be apparent to a skilled individual that attachment ofoligonucleotides to a solid material may be accomplished in a variety ofdifferent ways. For example, a number of different solid materials areavailable which have or can be attached or functionalised with asufficient density of functional groups in order to be useful for thepurpose of attaching or reacting with appropriately modifiedoligonucleotide probes. Further, a wide range of shapes, sizes and formsof such solid materials are available, including beads, resins,surface-coated plates, slides and capillaries. Examples of such solidmaterials used for covalent attachment of oligonucleotides include,without limitation: glass slides, glass beads, ferrite corepolymer-coated magnetic microbeads, silica micro-particles or magneticsilica micro-particles, silica-based capillary microtubes, 3D-reactivepolymer slides, microplate wells, polystyrene beads, poly(lactic) acid(PLA) particles, poly(methyl methacrylate) (PMMA) micro-particles,controlled pore glass resins, graphene oxide surfaces and functionalisedagarose or polyacrylamide surfaces. Polymers such as polyacrylamide havethe further advantage that a functionalised oligonucleotide can becovalently attached during the polymerisation reaction between monomers(e.g. acrylamide monomers) that is used to produce the polymer. Afunctionalised oligonucleotide is included in the polymerisationreaction to produce a solid polymer containing covalently attachedoligonucleotide. Such polymerisation represents a highly efficient meansof attaching oligonucleotide to a solid material with control over thesize, shape and form of the oligonucleotide-attached solid materialproduced.

Typically in order to attach an oligonucleotide probe to any such solidmaterials, the oligonucleotide is synthesised with a functional group ateither the 3′ or 5′ end; although functional groups may also be addedduring the oligonucleotide production process at almost any baseposition. A specific reaction may then be performed between thefunctional group(s) within an oligonucleotide and a functional group onthe relevant solid material to form a stable covalent bond, resulting inan oligonucleotide attached to a solid material. Typically such anoligonucleotide would be attached to the solid material by either the 5′or 3′ end. By way of example, two commonly used and reliable attachmentchemistries utilise a thiol (SH) or amine (NH3) group and the functionalgroup in the oligonucleotide. A thiol group can react with a maleimidemoiety on the solid support to form a thioester linkage, while an aminecan react with a succinimidyl ester (NHS ester) modified carboxylic acidto form an amide linkage. A number of other chemistries can also beused. As well as chemical conjugation of an oligonucleotide probe to asolid material, it is possible and potentially advantageous to directlysynthesise oligonucleotide probes on a solid material for use in theperformance of the method.

In other embodiments the second oligonucleotide probe is attached to amoiety that permits its attachment to a solid material. One strategy isto employ a method of affinity binding whereby a moiety that permitsspecific binding may be attached to the oligonucleotide probe tofacilitate its attachment to the relevant affinity ligand. This may beperformed, for example, using antibody-antigen binding or an affinitytag, such as a poly-histidine tag, or by using nucleic acid basedhybridisation wherein the complementary nucleic acid is attached to asolid material, e.g. a nitrocellulose nucleic acid lateral flow strip.An exemplary such moiety is biotin, which is capable of high affinitybinding to streptavidin or avidin which itself is attached to beads oranother solid surface.

The presence of two or more different target nucleic acids of definedsequence may be detected in the same sample. In an embodiment of themethod, separate series of steps a), b), and c), using differentoligonucleotide primers and oligonucleotide probes for each of the twoor more target nucleic acids is performed, which separate steps may beconducted simultaneously. For example, in an embodiment, one set ofoligonucleotide primers and oligonucleotide probes would be used for thedetection of one target nucleic acid in a sample and another set ofoligonucleotide primers and oligonucleotide probes would be used for thedetection of another target nucleic acid in the same sample. Thedetection of the detector species produced from the two or moredifferent sets of primers/probes could each be coupled to a particularsignal, such as different colorimetric or fluorometric dyes or enzymes,to allow multiplex detection. Alternatively multiplex detection may beaccomplished by the attachment of the second oligonucleotide probe to asolid material, directly or indirectly through a moiety that permits itsattachment to a solid material. Such an approach utilises physicalseparation of the detector species produced by the different series ofsteps a), b) and c), rather than relying on a different detection means.Thus, for example, a single dye could be used on nucleic acid lateralflow to detect multiple different target nucleic acids wherein eachdifferent detector species produced is localised to a particular printedline on the lateral flow strip and direct or indirect sequence basedhybridisation to the second oligonucleotide probe forms the basis of thedifferential detection. Alternatively an electrical detection array maybe used wherein multiple different second oligonucleotide probes areattached to a particular region of the array and thus in a multiplexreaction wherein multiple different detector species are produced at thesame time, each detector species becomes localised via hybridisation toa discrete region of the array permitting multiplex detection.

The foregoing detection processes, such as nucleic acid lateral flow andelectrical detection, and their ability to readily detect multipledifferent target nucleic acids within the same sample, are enabled bythe intrinsic requirement of the present method for two oligonucleotideprobes. As such they powerfully demonstrate the advantages of the methodof the invention over known methods.

The current invention is of broad utility to various fields andapplications which require detection of a target nucleic acid of definedsequence in a sample. It represents a fast, cheap and convenient meansof determination of the presence of a target nucleic acid sequencewithin a sample. By way of a list of applications that is in no waylimiting, we envisage that the invention could be of value in fieldssuch as; diagnostics, forensics, agriculture, animal health,environment, defence, human genetic testing, prenatal testing, bloodcontamination screening, pharmacogenomics or pharmacokinetics andmicrobiological, clinical and biomedical research. Suitably the sampleis a biological sample such as a human sample. The sample may be a humansample, a forensic sample, an agricultural sample, a veterinary sample,an environmental sample or a biodefence sample.

Detection of target nucleic acid may be used for the diagnosis,prognosis or monitoring of disease or a diseased state such as aninfectious disease, including but not limited to HIV, influenza, RSV,Rhinovirus, norovirus, tuberculosis, HPV, meningitis, hepatitis, MRSA,Ebola, Clostridium difficile, Epstein-Barr virus, malaria, plague,polio, chlamydia, herpes, gonorrhoea, measles, mumps, rubella, choleraor smallpox, or cancer, including but not limited to colorectal cancer,lung cancer, breast cancer, pancreatic cancer, prostate cancer, livercancer, bladder cancer, leukaemia, esophageal cancer, ovarian cancer,kidney cancer, stomach cancer or melanoma, or in the fields of humangenetic testing, prenatal testing, blood contamination screening,pharmacogenetics or pharmacokinetics.

The invention is amenable for use with a broad array of sample types,such as, for example: Nasal swabs or aspirates, nasopharyngeal swabs oraspirates, throat swabs or aspirates, cheek swabs or aspirate, blood ora sample derived from blood, urine or a sample derived from urine,sputum or a sample derived from sputum, stool or a sample derived fromstool, cerebrospinal fluid (CSF) or a sample derived from CSF, andgastric fluids or a sample derived from gastric fluids, human or animalsamples derived from any form of tissue biopsy or bodily fluid. We havealso performed the method in a broad range of samples containing atleast 10-20% of the following clinical specimens: Nasal swab in VTM,nasopharyngeal swab in VTM, thin prep media, throat swab in liquidAmies, HSV sore swab in M4 media, synovial fluid, sputum processed via2M NaOH/isopropanol followed by DNA capture beads, rectal swab in TE,stool sample processed by homogenisation and DNA capture beads, CSF,APTIMA swab, amniotic fluid, oral swab in liquid Amies, urine, VRE swabin TE, pleural fluid, whole blood, K2EDTA plasma, L.Heparin plasma andblood serum. These experiments have demonstrated the remarkableversatility of the method to different clinical applications and thelack of inhibition observed in relevant samples. This is in starkcontrast to other methods which are inhibited by inhibitors found inbiological specimens, such as heparin and phytic acid which inhibit PCR,and therefore demonstrates the potential to use the method in a low-costor single-use device without any requirement for complex samplepreparation procedures.

The target nucleic acid may be (a) viral or derived from viral nucleicacid material (b) bacterial or derived from bacterial nucleic acidmaterial (c) circulating, cell-free DNA released from cancer cells (d)circulating, cell-free DNA released from foetal cells or (e) micro RNAor derived from micro RNA inter alia.

The single stranded target nucleic acid for the method may be naturallyoccurring or non-naturally occurring. The target nucleic acid may begenerated in situ or produced from a naturally occurring nucleic acidprior to performance of the method. A single stranded target nucleicacid for the method may be prepared by one or more additional stepsperformed prior to or simultaneously with step a), which additionalsteps may encompass one or more enzymes such as polymerases andrestriction enzymes. Generating the target nucleic acid for the methodin this way has a number of potential advantages, such as permittingeven more highly multiplexed assays and/or overcoming ab initiobackground. A highly specific conversion of nucleic acid material in abiological sample may, for example, be performed without amplificationprior to amplification in step a). The sample may be, for example,treated, purified, subject to buffer exchange, subject to exome capture,partially depleted of contaminating material and/or converted to asingle stranded target nucleic acid for the method containing one ormore modified dNTP. Provided that the “real” target nucleic acid in thesample to be detected is converted into the “surrogate” target nucleicacid for performance of the method with reliable conversion (which maybe <1:1, 1:1 or 1:>1, i.e. possibly with some element of amplification)then detection of the “surrogate” target nucleic acid will allow the“real” nucleic acid to be detected and/or quantified. Furthermore,production of a surrogate target from a naturally occurring target inthis way can be used to generate in a specific manner a target nucleicacid for the method with any desired sequence. In an embodiment whereinthe single stranded target nucleic acid is derived from double strandedDNA following disassociation of the two strands, e.g. by strandinvasion, two complementary single stranded nucleic acid targets arepresent and may be amplified and detected in a reciprocal process by thesame oligonucleotide primers and probes. Wherein the target is thegenome of a −ve strand single stranded RNA virus, the +ve strandtranscript may also be present in the sample and either strand or bothstrands may be amplified and detected as the single stranded targetnucleic acid in the method using the same oligonucleotide primers andprobes.

It is also envisaged that the present invention has the potential to beof utility in screening samples for cell free DNA and epigeneticmodifications such as, for example, CpG methylation of DNA sequences.Such epigenetic modification of particular cancer associated targetgenes can serve as useful biomarkers in a number of diseases and diseasestates. Given the growing appreciation of the importance of epigeneticmodification in human disease, there is potential for the presentinvention to be used to specifically assess the epigenetic modificationof particular target nucleic acid biomarkers based upon the differentialactivity of the strand displacement DNA polymerase and/or restrictionenzymes. Therefore, in an embodiment, the target nucleic acid contains asite of epigenetic modification, such as methylation. Alternatively the“real” nucleic acid used to produce a “surrogate” target nucleic acidfor the performance of the method, as described above, contains a siteof epigenetic modification.

A further aspect of the invention relates to kits for use in thedetection of nucleic acids of defined sequence in a sample. Thus theinvention also provides a kit comprising the following:

-   -   a) a first oligonucleotide primer and a second oligonucleotide        primer wherein said first primer comprises in the 5′ to 3′        direction a restriction enzyme recognition sequence and cleavage        site and a region that is capable of hybridising to a first        hybridisation sequence in a single stranded target nucleic acid        of defined sequence, and said second primer comprises in the 5′        to 3′ direction a restriction enzyme recognition sequence and        cleavage site and a region that is capable of hybridising to the        reverse complement of a second hybridisation sequence upstream        of the first hybridisation sequence in the target nucleic acid;    -   b) a first restriction enzyme that is not a nicking enzyme and        is capable of recognising the recognition sequence of and        cleaving the cleavage site of the first primer and a second        restriction enzyme that is not a nicking enzyme and is capable        of recognising the recognition sequence of and cleaving the        cleavage site of the second primer;    -   c) a strand displacement DNA polymerase;    -   d) dNTPs;    -   e) one or more modified dNTP;    -   f) a first oligonucleotide probe which is capable of hybridising        to a first single stranded detection sequence in at least one        species in amplification product produced in the presence of        said target nucleic acid and which is attached to a moiety which        permits its detection; and    -   g) a second oligonucleotide probe which is capable of        hybridising to a second single stranded detection sequence        upstream or downstream of the first single stranded detection        sequence in said at least one species in amplification product        and which is attached to a solid material or to a moiety which        permits its attachment to a solid material.

In an embodiment one of the first and second oligonucleotide probes ofthe kit is blocked at the 3′ end from extension by the DNA polymeraseand is not capable of being cleaved by either the first or secondrestriction enzymes, for example due to the presence of one or moresequence mismatch and/or one or more modifications such as aphosphorothioate linkage.

In an embodiment one of the first and second oligonucleotide probes ofthe kit has 5 or more bases of complementarity to the hybridising regionor the reverse complement of the hybridising region of the first orsecond primer.

In another embodiment the first oligonucleotide probe of the kit hassome complementarity, e.g. 5 or more bases of complementarity, to thehybridising region of one of the first and second oligonucleotideprimers, and/or the second oligonucleotide probe of the kit has somecomplementarity, e.g. 5 or more bases of complementarity, to the reversecomplement of the hybridising region of the other of the first andsecond oligonucleotide primer.

In further embodiments the first and/or second oligonucleotide probesmay have some complementarity or reverse complementarity to the gapbetween the first and second hybridisation sequences in the targetnucleic acid as described above.

The kit may also comprise a reverse transcriptase.

The kit may additionally comprise means to detect the presence of adetector species produced in the presence of the target nucleic acid.For example, the kit may additionally comprise a nucleic acid lateralflow strip, an electrochemical probe a 96-well plate, beads or an arraysurface, and/or a colorimetric or fluorometric dye and/or a device forthe detection of a change in electrical signal, and/or carbon or gold.

In various embodiments the target nucleic acid and the components of thekits, such as, the first oligonucleotide primer and/or the secondoligonucleotide primer and/or the first restriction enzyme and/or thesecond restriction enzyme and/or the DNA polymerase and/or the dNTPsand/or the one or more modified dNTP and/or the first oligonucleotideprobe and/or the second oligonucleotide probe and/or the either thefirst or second single stranded detection sequence in the at least onespecies within the amplification product comprised in the kit are asdefined herein for the methods of the invention. For example, the kitmay comprise any combination of the features of such componentsdescribed herein, such as, without limitation, the following: One of thefirst and second oligonucleotide probes is blocked at the 3′ end fromextension by the DNA polymerase and is not capable of being cleavage byeither the first or second restriction enzymes optionally due to thepresence of one or more sequence mismatch and/or one or moremodifications such as a phosphorothioate linkage; the first restrictionenzyme and the second restriction enzyme are the same restrictionenzyme; the one or more modified dNTP is an alpha thiol modified dNTP;the moiety that permits the detection of the first oligonucleotide probeis a colorimetric or fluorometric dye or a moiety that is capable ofattachment to a colorimetric or fluorometric dye, such as biotin; themoiety that permits the attachment of the second oligonucleotide probeto a solid material is a single stranded oligonucleotide, optionallycomprising three or more repeat copies of a 2 to 4 based DNA sequencemotif; the first and second oligonucleotide primers comprise astabilising sequence upstream of the restriction enzyme recognitionsequence and cleavage site, such as at the 5′ end, and e.g. of 5 or 6bases in length; the hybridising region of the first and/or secondoligonucleotide primers is between 6 and 30, e.g. 9 and 16, bases inlength; and, the first and second hybridisation sequences in the targetnucleic acid are separated by 0 to 15 or 0 to 6 bases, in certainembodiments they are separated by 3 to 15 or by 3 to 6 bases, e.g. 5, 7or 11 bases, or they are overlapping such as by 1 to 2 bases.

The kits may comprise means to detect the presence of a detector speciesproduced in the presence of the target nucleic acid, such as a nucleicacid lateral flow strip. In a further embodiment, the kit additionallycomprises the third and/or fourth oligonucleotide primers as definedherein.

The kits may also include reagents such as reaction buffers, salts e.g.divalent metal ions, additives and excipients.

The kits according to the invention may be provided together withinstructions for the performance of the methods according to theinvention.

The invention also provides the use of the kits of the invention for thedetection of a single stranded target nucleic acid of defined sequencein a sample.

It is to be understood that all the optional and/or preferredembodiments of the invention described herein in relation to the methodsof the invention also apply in relation to the kits of the invention andthe use thereof, and vice versa.

As mentioned previously the methods and kits of the invention areideally suited for use in a device, such as a single-use diagnosticdevice. Thus the invention also provides a device containing a kit asdescribed above, in particular a kit comprising means to detect thepresence of a detector species produced in the presence of the targetnucleic acid, such as a nucleic acid lateral flow strip. The device maybe a powered device, e.g. an electrically powered device, the device mayalso comprise heating means and may be a self-contained device, i.e. adevice that requires no ancillary test instrument.

The method of the invention may also be used independently from thedetection step c) for amplifying a nucleic acid signal from a targetnucleic acid of defined sequence, such a method may be used, forexample, if the amplified signal is to be stored and/or transported fordetection of the target nucleic acid at a future date and/or alternativelocation if required. The amplified signal comprises the pre-detectorspecies or detector species produced through performance of the method.Thus in a further embodiment the invention provides a method ofamplifying a nucleic acid signal from a target nucleic acid of definedsequence in a sample comprising steps a) and all or part, e.g. part i.or ii., of step b) of the method of the invention.

The invention also provides the use of the kits of the invention foramplifying a nucleic acid signal from a target nucleic acid of definedsequence as defined above.

It is to be understood that all the optional and/or preferredembodiments of the invention described herein in relation to the methodsof the invention for detecting the presence of a target nucleic acid ofdefined sequence in a sample also apply in relation to the method foramplifying a nucleic acid signal from a target nucleic acid of definedsequence.

The following examples serve to further illustrate various aspects andembodiments of the methods described herein. These examples should notbe considered limiting in any way.

EXAMPLES Materials and Methods

The following materials and methods are used in the examples belowunless otherwise indicated.Oligonucleotides: Except as otherwise indicated custom oligonucleotideswere manufactured using the phosphoramidite method by Integrated DNATechnologies.Nucleic Acid Lateral Flow: Carbon nanoparticles were conjugated vianon-covalent adsorption to various biotin-binding proteins, e.g.streptavidin. Typically, a colloidal carbon suspension was prepared inBorate Buffer followed by sonication using a probe sonicator. Carbon wassubsequently adsorbed to biotin-binding protein by incubation at roomtemperature. Carbon was either used directly in the reaction mixtures orapplied to glass fibre conjugate pads. Lateral flow strips wereconstructed by combining a conjugate pad containing lyophilised sugarsand additives used to improve visual appearance with a sample pad,nitrocellulose membrane and adsorbent pad (Merck Millipore) followingthe manufacturer's guidelines. Prior to its use in lateral flow strips,the relevant oligonucleotide(s) containing the reverse complement of thesequence to be detected in the method were printed onto thenitrocellulose membrane at a defined location and attached to themembrane via UV cross-linking.

Example 1 Performance of the Method Wherein the Second OligonucleotideProbe is Attached to a Solid Material, a Nitrocellulose Lateral FlowStrip

This example demonstrates the performance of the method wherein thesecond oligonucleotide probe is attached to a solid material, anitrocellulose lateral flow strip, and the first oligonucleotide probeis not contacted with the sample simultaneously to the performance ofthe amplification step a).

The first oligonucleotide primer with a total length of 24 bases wasdesigned comprising in the 5′ to 3′ direction: A stabilising region of 7bases; the 5 bases of the recognition sequence for a restriction enzymethat is not a nicking enzyme; and a 12 base hybridising regioncomprising the reverse complementary sequence of the first hybridisationsequence in the target nucleic acid. The second oligonucleotide primerwas designed to contain the same stabilising region and restrictionenzyme recognition sequence, but with the 12 base hybridising regioncapable of hybridising to the reverse complement of the secondhybridisation sequence in the target nucleic acid. In this example thefirst restriction enzyme and the second restriction enzyme are the samerestriction enzyme. The restriction enzyme is an asymmetricdouble-strand cleaving restriction enzyme with a top strand cleavagesite downstream of its 5 base recognition sequence. The first and secondhybridisation sequences in the target nucleic acid are separated by 1base.

The oligonucleotide primers were designed using the target nucleic acid,such that the nucleotide base downstream of the cleavage site in thereverse complement of the primers is Adenosine such that alpha thioldATP is employed as the modified dNTP in the method. A phosphorothioatemodification is inserted by the strand displacement polymerase to blockcleavage of said reverse complementary strand.

The first oligonucleotide probe with a total length of 20 bases wasdesigned comprising in the 5′ to 3′ direction: A 12 base region ofcomplementarity to at least one species in the amplification product; aneutral spacer region of 6 bases; and a 3′ biotin modification addedduring synthesis wherein said biotin modification permits attachment ofthe first oligonucleotide probe to a colorimetric dye, carbonnanoparticles. Carbon adsorbed to a biotin binding protein was preparedand saturated with the first oligonucleotide probe. The secondoligonucleotide probe with a total length of 49 bases was designed tocomprise, in the 5′ to 3′ direction: A neutral spacer comprising 10×Thymidine bases; 3× repeats of a 13 base region capable of hybridisingto the second single stranded detection sequence downstream of the firstsingle stranded detection sequence in said at least one species in theamplification product. Approximately 30 pmol of said secondoligonucleotide probe was printed on the nucleic acid flow strip.

Reactions were prepared containing; 1.6 pmol of the first primer; 0.1pmol of the second primer; 250 μM2′-Deoxyadenosine-5′-O-(1-thiotriphosphate) Sp-isomer (Sp-dATP-α-S) fromEnzo Life Sciences; 60 μM of each of dTTP, dCTP and dGTP; 2U of therestriction enzyme; and 2U of a Bacillus strand displacement DNApolymerase. The nucleic acid target (a single stranded DNA target) wasadded at various levels (++=1 amol, +=10 zmol, NTC=no target control) ina 10 μl total reaction volume in an appropriate reaction buffer.Reactions were incubated at 45° C. for 7 min or 10 min. 6.5 μl of theterminated reaction mix was then added to 60 μl lateral flow runningbuffer containing 0.056 mgml⁻¹ of the conjugated carbon before beingloaded onto the nucleic acid lateral flow strip with the secondoligonucleotide probe attached to it in a printed line.

FIG. 5 displays a photograph of the lateral flow strips obtained in theperformance of the example. An arrow indicates the position where thesecond oligonucleotide probe has been printed on the nitrocellulosestrip and hence where positive signal appears. A clear black linecorresponding to the presence of the carbon signal was observed only inthe presence of the target nucleic acid at both target levels and atboth time points demonstrating the rapid and sensitive detection of thetarget nucleic acid sequence by the method of the invention.

Example 2 Performance of the Method Wherein the First OligonucleotideProbe is Blocked at the 3′ End from Extension by the DNA Polymerase andis not Capable of being Cleaved by Either the First or SecondRestriction Enzyme and is Contacted with the Sample in Step a)

This example demonstrates the performance of embodiments of the methodswherein the first oligonucleotide probe is blocked at the 3′ end fromextension by the DNA polymerase and is not capable of being cleaved byeither the first or second restriction enzyme and contacted with thesample simultaneously to the performance of step a). In suchembodiments, we have not observed any significant inhibition of the rateof the amplification, indicating that the pre-detector speciesaccumulates in real-time without disrupting the optimal cyclicalamplification process. Not only have we not observed any inhibitoryeffects on the amplification process in said embodiments but we haveobserved a surprising enhancement of the signal produced correspondingto an increased amount of detector species, of at least 100-fold.

Example 2.1: A variant of the assay used in Example 1 was designedexploiting the embodiment of the method wherein the firstoligonucleotide probe is blocked at the 3′ end from extension by the DNApolymerase and is not capable of being cleaved by either the first orsecond restriction enzyme and contacted with the sample simultaneouslyto the performance of step a). The same oligonucleotide primers,restriction enzyme, dNTPs, modified dNTP and polymerase as employed inExample 1 were used, however, an alternative first oligonucleotide probewas designed with a total length of 21 bases comprising in the 5′ to 3′direction: A 5′ biotin modification; a neutral region of 8 bases; a 13base region capable of hybridising to at least one species in theamplification product; and a 3′ phosphate modification, wherein thebiotin modification permits attachment of the first oligonucleotideprobe to a colorimetric dye, carbon nanoparticles, and the phosphatemodification blocks its extension by the strand displacement DNApolymerase. Carbon adsorbed to a biotin binding protein was prepared andsaturated with the first oligonucleotide probe.

An alternative second oligonucleotide probe was designed with a totallength of 51 bases comprising, in the 5′ to 3′ direction: A 14 baseregion capable of hybridising to the second single stranded detectionsequence upstream of the first single stranded detection sequence insaid at least one species in the amplification product; a 6 base neutralspacer sequence; a repeat of the 14 base hybridising region; a second 6base neutral spacer sequence; and a 10× Thymidine base spacer.Approximately 30 pmol of said second oligonucleotide probe was printedon the nucleic acid flow strip.

Reactions were prepared containing: 0.8 pmol of the first primer; 0.8pmol of the second primer; 0.6 pmol of the first oligonucleotide probe;300 μM Sp-dATP-α-S; 60 μM of each of dTTP, dCTP and dGTP; 2U of therestriction enzyme; and 2U of a Bacillus strand displacement DNApolymerase. The nucleic acid target (a single stranded DNA target) wasadded at various levels (++=1 amol, +=10 zmol, NTC=no target control) ina 10 μl total reaction volume in an appropriate reaction buffer.Reactions were incubated at 45° C. for 6 min. 5 μl of the terminatedreaction mix was then added to 60 μl lateral flow running buffercontaining 0.03 mgml⁻¹ conjugated carbon before being loaded onto thenucleic acid lateral flow strip. A control reaction was performed inorder to demonstrate that no detector species is produced where no firstoligonucleotide probe was present during the reaction. The equivalentlevel (0.6 pmol) of the probe was added to said control after step a) inorder to control for any unintended impact of the presence of the probeduring the lateral flow strip detection.

FIG. 6A presents a photograph of the nucleic acid lateral flow stripsfollowing their development. Clear signal corresponding to deposition ofthe carbon nanoparticles was observed at both target levels when thefirst oligonucleotide probe was provided during the reaction. Asexpected, no signal was detected at either target level when the firstoligonucleotide was not provided during the reaction. This experimentdemonstrates clearly the potential to substantially enhance theproduction of the detector species in embodiments of the method whereinthe first oligonucleotide probe is blocked at the 3′ end from extensionby the DNA polymerase and is not capable of being cleaved by either thefirst or second restriction enzyme and contacted with the samplesimultaneously to the performance of step a). It is noteworthy that, incontrast to Example 1, an equal concentration of the first and secondoligonucleotide primers was provided, which enables more rapidamplification.

Example 2.2: A separate assay was next designed to demonstrate theversatility of the said embodiments of the method with an entirelydifferent target nucleic acid. The oligonucleotide primers andoligonucleotide probes were designed for the relevant target nucleicacid, a single stranded DNA, in a similar manner as described inExamples 1 and 2.1.

Reactions were prepared containing; 0.8 pmol of the first primer; 0.4pmol of the second primer; 0.6 pmol of the first oligonucleotide probe;300 μM Sp-dATP-α-S; 60 μM of each of dTTP, dCTP and dGTP; 2U of therestriction enzyme; and 2U of a Bacillus strand displacement DNApolymerase. The nucleic acid target (a single stranded DNA target) wasadded at various levels (+=1 amol, NTC=no target control) in a 10 μltotal reaction volume in an appropriate reaction buffer. Reactions wereincubated at 45° C. for 6 min. 5 μl of the terminated reaction mix wasthen added to 60 μl lateral flow running buffer containing 0.08 mgml⁻¹conjugated carbon before being loaded onto the nucleic acid lateral flowstrip. A control reaction was performed comprising a truncated variantof the first oligonucleotide probe that was also contacted with thesample simultaneously to the performance of step a).

FIG. 6B presents a photograph of the nucleic acid lateral flow stripsfollowing their development. Clear positive signal was visible in thepresent of the target nucleic acid and not in the no target controldemonstrating the correct design and functioning of the assay and therobust potential of the embodiments of the method wherein the firstoligonucleotide probe is blocked at the 3′ end from extension by the DNApolymerase and is not capable of being cleaved by either the first orsecond restriction enzyme and contacted with the sample simultaneouslyto the performance of step a). As expected only a very minimal signalwas observed in the control assay employing a truncated form of thefirst oligonucleotide probe, demonstrating the requirement for correcthybridisation of the first oligonucleotide probe simultaneously to theperformance of the amplification in step a) for the efficient productionof the detector species.

Example 3 Performance of the Method Wherein the Presence of Two or MoreDifferent Target Nucleic Acids of Defined Sequence are Detected in theSame Sample

This example demonstrates the potential of the method for the detectionof two or more different target nucleic acids of defined sequence in asample. The use of two oligonucleotide probes in addition to the primersin the method, provides an integral approach for detection of theamplification product in the method that is ideally suited to thedetection of two or more different target nucleic acids in the samesample. In this example the ability to differentially detect alternativedetector species based on the sequence specific hybridisation of thesecond oligonucleotide probe is demonstrated.

Firstly, in order to demonstrate the ability of the method to beemployed for the detection of two or more different target nucleic acidswe developed compatible sets of oligonucleotide primers and probes fordetection of two distinct targets (A and B). In each case the firstoligonucleotide probe was designed to contain the following features inthe 5′-3′ direction: a 5′ Biotin modification, a 7 base stabilisingregion, the 5 bases of a restriction endonuclease recognition site, a11-13 base region complementary to the 3′ end of the target A or Bcomprising a phosphorothioate bond at the cleavage site for therestriction enzyme, and a 3′ phosphate modification. The secondoligonucleotide probes were designed to contain in the 5′-3′ direction:A 12-14 base region complementary to the 5′ end of the target A or B, aneutral spacer of 5× Thymidine bases, and a single strandedoligonucleotide moiety of 12 bases as the moiety permitting theattachment of the second oligonucleotide probe to a solid material. Thesequence of the single stranded oligonucleotide attachment moiety foreach target was designed using a different sequence in order to permitthe attachment of each detector species to a different location on thelateral flow strip. Nucleic acid lateral flow strips were preparedcontaining discrete spots of 30 pmol of an oligonucleotide containingthe reverse complementary sequence to each single strandedoligonucleotide detection moiety at separate locations.

Reactions were assembled containing: 0.5 pmol of the firstoligonucleotide probe for target A and target B; 0.5 pmol of the secondoligonucleotide primer for target A and B, in 65 μl of an appropriatebuffer containing 0.032 mgml⁻¹ carbon adsorbed to a biotin bindingprotein. Different levels of each target (+=0.1 pmol; ++=1 pmol) wereadded to separate reactions individually and both targets were addedtogether. A no target control (NTC) was also performed.

FIG. 7A displays a photograph of the lateral flow strips obtained in theexperiment. Clear black spots corresponding to the deposition of thecarbon containing detector species were observed at both target levelsand for both assays. Furthermore when both reactions were performed atthe same time, the signal corresponding to both targets A and B wasobserved. No background signal or cross-talk between the differentassays was observed.

In order to demonstrate the robustness of the method, a furtherexperiment went on to develop three separate assays to demonstrate thepotential of the method for the detection of three different targetnucleic acids of defined sequence in a sample. A similar methodology wasemployed as described above. FIG. 7B displays a photograph of thelateral flow strips obtained. The targets P1, P2 and P3 were addedindividually and in various combinations as indicated. The reversecomplement to the single stranded oligonucleotide detection moiety ofthe second oligonucleotide probe was printed on the nucleic acid lateralflow strip in separate lines. The black signal indicates the depositionof the carbon attached detector species localised to the expectedlocation in all cases for rapid sensitive detection with no unintendedcross-talk between the assay nor any background signal. An equivalentexperiment comprising four separate assays demonstrates the potential ofthe method for the detection of four different target nucleic acids (P1,P2, P3 and P4) of defined sequence in a sample with the resultsdisplayed in FIG. 7C. In this four-target experiment, P4 was present inall reactions as a positive control and the other targets were addedindividually to separate reactions. The photographs of the lateral flowstrips displayed reveal clear black bands at the expected locations,corresponding to the presence of the relevant detector species bound tocarbon. Such multiplex assays demonstrate the potential of the method tobe used for diagnostic tests for diseases that are caused by a number ofdifferent pathogens wherein detecting the presence of the detectorspecies of the control assay indicates that the method has beenperformed successfully and the visualisation of one or more of the otherdetector species on the lateral flow strip indicates the presence of therelevant causative pathogen(s) in an appropriate clinical specimen.Whilst it would be rare in such diagnostic applications, such as in thefield of infectious diseases, to observe co-infections wherein more thanone pathogen is present at the same specimen, the method of theinvention is highly versatile for any combination of the targets in amultiplex reaction to be detected. FIG. 7D, displays the results of anexperiment wherein different combinations of four targets (P1, P2, P3and P4) are added. The ability to detect each target individually andthe detect the other three targets when each target is omitted withoutnon-specific background demonstrates the remarkable specificity ofdetection of the method of the invention.

In the above described and various other experiments, we have alsoperformed multiplex assays for the detection of 3-5 targets at very lowtarget concentrations, e.g. 1 zmol (600 copies) or 17 ymol (10 copies).In this example, we have clearly demonstrated the potential of themethod to detect the presence of two or more different target nucleicacids of defined sequence in a sample, and its potential for rapid,low-cost signal detection, e.g. by nucleic acid lateral flow. It is anunusual and advantageous feature of the method of the invention that twoor more different target nucleic acids of defined sequence can bereadily detected in the same sample. For each additional target to bedetected, an additional set of oligonucleotide primers is required,which in prior art methods without temperature cycling presents asignificant challenge to detecting the presence of two or more differenttarget nucleic acids, because the additional primers lead to anincreased propensity to form non-specific amplification products. In themethod of the invention, this challenge is overcome by specificityenhancement, such as that resulting from the use of modified bases,improved enzyme selection and the formation of a detector species usingthe oligonucleotide probes that exploit additional sequence specifichybridisation events.

Example 4 Performance of the Method Wherein the First and SecondHybridisation Sequences in the Target Nucleic Acid are Separated by 5Bases

This example demonstrates the performance of the method wherein thefirst and second hybridisation sequences in the target nucleic acid areseparated by 5 bases. The ability to use the target derived sequencethat is not present in the oligonucleotide primers and is only producedin the amplification product in a target dependent manner when the twooligonucleotide primers are designed to have a gap between the first andsecond hybridisation sequences, provides the potential for enhancedspecificity in embodiments of the method that can overcome anybackground signal arising from ab initio synthesis or primer-primerbinding. In said embodiments the sequence specific hybridisation of thefirst or second oligonucleotide probe is designed to exploit the gapbetween the two hybridisation regions in order that the detector speciesis only produced when the amplification product contains the correcttarget derived sequence.

In this example we designed a range of assays to demonstrate thehybridisation of the second oligonucleotide probe to various differentamplification products that differ only in the sequence of the gapbetween the first and second hybridisation sequences within the targetnucleic acid. The second oligonucleotide probe was designed to containan 11 base hybridising region for the at least one species in theamplification product at its 5′ end. Said region was made of up of a 7base sequence that is the reverse complementary sequence of the firstoligonucleotide primer and a 5 base sequence that is reversecomplementary sequence to additional target derived sequence in theamplification product derived from the gap between the two primers. Thesecond oligonucleotide probe also contained in the 5′ to 3′ direction aneutral spacer of 5× thymidine bases and a 12 base single strandedoligonucleotide moiety for its attachment to a solid material. Anitrocellulose nucleic acid flow strip printed with 30 pmol of anoligonucleotide with the reverse complementarity sequence of said moietywas prepared. The first oligonucleotide probe was designed to containthe same sequence as the second oligonucleotide primer but with a 5′biotin modification, a 3′ phosphate modification and a phosphorothioateinternucleotide linkage at the position of the restriction enzymecleavage site.

Four different artificial target nucleic acid sequences (T1, T2, T3 andT4) were designed, each of which had the exact sequence corresponding tothe first and second hybridisation sequences, but which differed in thefive bases between the first and second hybridisation sequences: T1contains the correct bases for detection with full complementarity tothe 11 base hybridising region of the second oligonucleotide probe; T2contains four mismatches out of the five bases of the gap; T3 wasdesigned so that four bases out of the five bases of the gap are removedand therefore the species of the amplification product are four basesshorter. T4 contains two mismatches out of the five bases of the gap.

Reactions were assembled containing: 3.6 pmol of the firstoligonucleotide primer; 1.8 pmol of the second oligonucleotide primer;2.4 pmol of the first oligonucleotide probe; 30004 Sp-dATP-α-S, 60 μMdTTP, dCTP, dGTP; 12U Restriction enzyme; 12U of a Bacillus stranddisplacement DNA polymerase in a total reaction volume of 60 μl in anappropriate reaction buffer. 1 amol target (T1, T2, T3 or T4) was addedto each reaction before incubation at 45° C. for 6.5 min before 53.5 μlof the 60 μl reaction was run on the lateral flow strip. Prior toapplication of the reaction to the lateral flow strip, 1.5 pmol of thesecond oligonucleotide probe and 2 μg carbon adsorbed to biotin bindingprotein were deposited onto the conjugate pad and left to dry for 5 min.

FIG. 8 displays a photograph of the nucleic acid lateral flow stripsobtained in the experiment. The strip obtained with target T1 shows aclear black line corresponding to carbon attached detector speciesattached to the solid material of the nitrocellulose and evidencing thatthe assay developed in this example including the oligonucleotideprimers and probes functions correctly and has the potential for rapidand sensitive detection. Reactions performed with targets T2 and T3 didnot reveal any carbon corresponding to positive signal, evidencing thatboth four mismatches and the removal of four bases removes the abilityfor the second oligonucleotide to hybridise effectively to thepre-detector species produced in the reaction. A very faint signal wasobserved on the strip produced using T4 indicating that the presence ofonly two mismatch bases leads to a substantial loss in the ability ofthe second oligonucleotide probe to successfully hybridise to thepre-detector species to product the detector species capable of bindingto the line on the strip. Polyacrylamide gel electrophoresis wasperformed using repeat reactions to confirm that all reactions with alltargets functioned correctly and produced a significant amount ofamplification product. An expected a size shift was visible in thereaction performed with the four base truncated target T3.

This example demonstrates how the first and second oligonucleotideprobes, an integral feature of the present invention, provide not onlyfor the rapid and sensitive detection of the amplification product, butcan also be used to provide a further target sequence based specificitycheck on the amplification product beyond that resulting from primerhybridisation alone. This powerful technique overcomes the knownproblems of prior art methods resulting from non-target specificbackground amplification in certain assays resulting from ab initiosynthesis or primer-primer binding. It demonstrates the method of theinvention exhibits enhanced specificity compared to prior art methods,whilst retaining sensitive detection and rapid, low-cost resultsvisualisation.

Example 5 Performance of the Method Wherein the Moiety that Permits theAttachment of the Second Oligonucleotide Probe to a Solid Material is anAntigen and the Corresponding Antibody is Attached to a Solid Surface, aNitrocellulose Lateral Flow Strip

In the method of the invention, a number of different moieties may beemployed as the moiety for the attachment of the second oligonucleotideprobe to a solid material. This example demonstrates that the method canbe performed wherein the moiety that permits the attachment of thesecond oligonucleotide probe to a solid material is an antigen and thecorresponding antibody is attached to a solid surface, a nitrocelluloselateral flow strip.

A second oligonucleotide probe was designed to comprise a 32 basesequence comprising a region of homology to at least one species in anamplification product and a 3′ Digoxigenin NHS Ester modification whichwas added during synthesis. A Fab fragment anti-digoxigenin antibodypurified from sheep (Sigma-Aldrich) was immobilised onto a nucleic acidlateral flow strip by spotting and air drying.

The performance of the second oligonucleotide probe was demonstrated inan experiment wherein various levels of the target (+++=1 pmol; ++=0.1pmol; +=10 fmol; NTC=no target control) were added to 60 μl of acontrived reaction buffer containing the necessary reagents fordetection using a carbon nucleic acid lateral flow reaction, including0.016 mgml⁻¹ of carbon adsorbed to biotin binding protein. The strip wasprepared with 0.5 μg of anti-digoxigenin Fab fragment spotted onto thestrip in 0.2 μl buffer containing 2.5 mM Borate and 0.5% Tween 20. Thesolution was allowed to dry into the nitrocellulose membrane of thelateral flow strip for 2 h. Reactions were incubated at 45° C. for 2 minto form the contrived detector species before the entire reaction mix ofeach reaction was applied to a lateral flow strip.

FIG. 9 displays a photograph of the lateral flow strips produced in theexperiment. Black spots corresponding to the deposition of carbon on thelateral flow strip are visible at each target level and not visible inthe NTC indicating the specific detection of the detector species. Acombination of a biotin based affinity interaction for attachment of thedetection moiety (carbon) and an antibody based affinity interaction forsolid material attachment moiety has been demonstrated. This exampleserves to demonstrate the versatility of the method in terms ofdifferent approaches available for the attachment of the secondoligonucleotide probe to a solid material.

Example 6 Performance of the Method Wherein the Moiety that Permits theAttachment of the Second Oligonucleotide Probe to a Solid Material is aSingle Stranded Oligonucleotide Comprising Four Repeat Copies of a ThreeBase DNA Sequence Motif and the Reverse Complement of Said SingleStranded Oligonucleotide Sequence is Attached to a Solid Material

This example demonstrates the performance of the method wherein themoiety that permits the attachment of the second oligonucleotide probeto a solid material is a single stranded oligonucleotide comprising fourrepeat copies of a three base DNA sequence motif. As described above,embodiments of the method employing a single stranded oligonucleotide asthe detection moiety of the second oligonucleotide probe presents astraightforward and versatile aspect of the method, which facilitatesdetection by nucleic acid lateral flow and readily enables the detectionof multiple different target nucleic acids in the same sample. Further,the single stranded oligonucleotide detection moieties may be defined inadvance and optimised for efficient on-strip hybridisation to enhancethe sensitivity of detection and provide for efficient scale-upmanufacture of the nucleic acid lateral flow strip.

In one aspect of the invention we observed a surprising improvement tothe on-strip hybridisation by use of a single stranded oligonucleotidedetection moiety comprised of multiple repeat copies of a DNA sequencemotif. This example presents the results of multiple side-by-sideexperiments wherein the performance of an assay with the secondoligonucleotide attached directly to the lateral flow strip issubstantially enhanced by the use of a single stranded detection moietycomprising four repeat copies of a three base DNA sequence motif andwherein the reverse complement of said single stranded oligonucleotidesequence is attached to the lateral flow strip.

Example 6.1: An assay was designed exploiting the embodiment of themethod wherein the first oligonucleotide probe is blocked at the 3′ endfrom extension by the DNA polymerase and is not capable of being cleavedby either the first or second restriction enzyme and contacted with thesample simultaneously to the performance of step a). A firstoligonucleotide probe was designed with a total length of 25 basescomprising in the 5′ to 3′ direction: A 5′ Biotin modification; aneutral region of 7 bases; the 5 bases of a restriction enzymerecognition site that is not a nicking enzyme; a 13 base region capableof hybridising to the first hybridisation region in the targetcomprising a phosphorothioate bond at the cleavage site for therestriction enzyme; and a 3′ phosphate modification, wherein the biotinmodification permits attachment of the first oligonucleotide probe to acolorimetric dye, carbon nanoparticles, and the phosphate modificationblocks its extension by the strand displacement DNA polymerase.

Two alternative second oligonucleotide probes were designed to detectthe same target species (I and II). The second oligonucleotide probe ‘I’was designed to contain in the 5′ to 3′ direction: 3× repeats of a 14base region capable of hybridising to the reverse complement of thesecond hybridisation sequence in the target; and a 9× Thymidine basespacer. Nucleic acid lateral flow strips were prepared with spotscontaining 30 pmol of the probe.

The alternative second oligonucleotide probe ‘II’ was designed tocontain in the 5′-3′ direction: A 14 base region capable of hybridisingto the reverse complement of the second hybridisation region in thetarget; a neutral spacer of 5× Thymidine bases; and a single strandedoligonucleotide moiety of 12 bases comprising 4× repeat of a 3 basesequence motif which acts as the moiety permitting the attachment of thesecond oligonucleotide probe to a solid material. An additional singlestranded oligonucleotide was designed comprising in the 5′ to 3′direction: an 11× Thymidine base spacer; a 36 base region comprising a12× repeat of the reverse complement to the 3 base sequence motif whichforms the moiety permitting attachment of the second oligonucleotide IIto a solid material. For the second oligonucleotide probe II nucleicacid lateral flow strips were prepared spotted with 30 pmol of saidadditional single stranded oligonucleotide.

Reactions to test the performance of the oligonucleotide probes I and IIwere performed containing: 0.5 pmol of the first oligonucleotide probein 60 μl of an appropriate buffer containing 0.016 mgml-1 carbonadsorbed to biotin binding protein. Reactions for II were assembled inthe same manner but with the addition of 0.5 pmol of the secondoligonucleotide probe II. The nucleic acid target (a single stranded DNAtarget representative of at least one species within the amplificationproduct resulting from the designed assay reagents) was added at variouslevels (+++=1 pmol, ++=0.1 pmol, NTC=no target control). Assembledreactions were incubated for 2 min at 45° C. before the entire reactionmix was loaded onto the appropriate nucleic acid lateral flow strip.

FIG. 10A displays a photograph of the lateral flow strips obtained inthe experiment, with the left panel displaying results with secondoligonucleotide probe I and the right panel displaying results withsecond oligonucleotide probe II. Black spots corresponding to thedeposition of carbon attached detector species were visualised in thepresence of target. For the second oligonucleotide probe II comprisingthe repeat sequence motif a stronger signal was observed at all targetlevels.

Example 6.2: A separate assay was next designed for an entirelydifferent target nucleic acid to demonstrate the versatility of the saidembodiments of the method and its broad applicability. Theoligonucleotide probes were designed for the relevant target nucleicacid, a single stranded DNA, in a similar manner to that described inExample 6.1; again with two versions of the second oligonucleotide probereferred to as ‘I’ and ‘II’ and various target levels (+++=1 pmol,++=0.1 pmol, +=0.001 pmol). An even more striking effect was observed asdisplayed in the photograph of the lateral flow strips produceddisplayed in FIG. 10B. At the lower two target levels tested the secondoligonucleotide probe I did not produce any signal whereas thecorresponding repeat sequence oligonucleotide probe II produced a clearpositive signal indicated by the black spots of deposited carbon.

This example reveals a striking improvement to lateral flowhybridisation based detection employing a second oligonucleotidedetection moiety comprising repeat copies of a DNA sequence motif. Itdemonstrates that an improvement to the sensitivity of the nucleic acidlateral flow based detection of the detector species of 100-fold can beobtained. The intensity of the signal is enhanced and the signaldevelops more rapidly, demonstrating the potential for said embodimentsof the invention to be readily applicable to applications involvingrapid detection, such as by nucleic acid lateral flow. Furthermore thepotential of using a single stranded oligonucleotide as the detectionmoiety attached to the second oligonucleotide probe is exemplified.

Example 7 Use of the Method for the Detection of an RNA Virus inClinical Specimens

This example demonstrates the performance of the method to detect an RNAvirus in clinical specimens, using the embodiment of the method whereinthe first oligonucleotide probe is contacted with the samplesimultaneously to the performance of the amplification step a) and themoiety that permits the attachment of the second oligonucleotide probeto a solid material is a single stranded oligonucleotide comprising offour repeat copies of a three base DNA sequence motif and the reversecomplement of said single stranded oligonucleotide sequence is attachedto a solid material. In various investigations we have routinelydetected very low copies of RNA targets, such as viral genome extracts.For example, using quantified viral genome extracts we have employed themethod of the invention to detect less than 100 genome equivalent copiesof a virus in under 10 min total time to result, with an amplificationstep a) of less than 5 min. This remarkable rate and sensitivitydemonstrates the potential of the method for application in the field ofdiagnostics. As such, in this example, we have developed an assay todetect a pathogenic single stranded RNA virus and demonstrated theperformance of that assay using clinical specimens infected with thevirus.

The first oligonucleotide primer with a total length of 25 nucleotidebases was designed comprising in the 5′ to 3′ direction: A stabilisingregion of 8 bases synthesised to contain phosphorothioate bonds betweeneach base; the 5 bases of a recognition site for a restriction enzymethat is not a nicking enzyme; and a 12 base hybridising regioncomprising the reverse complementary sequence of the first hybridisationsequence in the target nucleic acid, designed to target a region withinthe single stranded RNA virus genome. The second oligonucleotide primerwas designed to contain the same stabilising region but without thephosphorothioate bonds and the same restriction enzyme recognitionsequence, but with the 12 base hybridising region capable of hybridisingto the reverse complement of the second hybridisation sequence. In thisexample the first restriction enzyme and the second restriction enzymeare the same restriction enzyme. The first and second hybridisationsequences in the target nucleic acid are separated by 0 bases.

The oligonucleotide primers were designed using the target nucleic acid,such that the nucleotide base downstream of the cleavage site in thereverse complement of the primers is Adenosine such that alpha thioldATP is employed as the modified dNTP for use in the method. Aphosphorothioate modification is inserted by the strand displacement DNApolymerase, or the reverse transcriptase to block cleavage of saidreverse complementary strand.

The first oligonucleotide probe with a total length of 24 bases wasdesigned comprising in the 5′ to 3′ direction: A 5′ Biotin modificationadded during synthesis wherein said biotin modification permitsattachment of the first oligonucleotide probe to a colorimetric dye,carbon nanoparticles, a stabilising region of 8 bases; the 5 bases ofthe recognition sequence for a restriction enzyme that is not a nickingenzyme wherein the cleavage site for said restriction enzyme in thefirst oligonucleotide probe is protected by a phosphorothioateinternucleotide linkage added during synthesis; an 11 base regioncapable of hybridising to at least one species in the amplificationproduct; and a 3′ phosphate modification which prevents extension by thestrand displacement DNA polymerase.

The second oligonucleotide probe with a total length of 31 bases wasdesigned comprising in the 5′ to 3′ direction: a 14 base region capableof hybridising to the second single stranded detection sequencedownstream of the first single stranded detection sequence in said atleast one species in the amplification product; a spacer comprising 5×Thymidine bases; 4× repeats of a three base DNA sequence motif, thereverse complement to which is immobilised on the lateral flow strip.The immobilised lateral flow printed oligonucleotide with a total lengthof 47 bases is designed comprising: A neutral spacer comprising 11×Thymidine bases; a 12× repeat of a 3 base sequence motif, which iscomplementary to the 3 base sequence motif of the second oligonucleotideprobe. A lateral flow control oligonucleotide with a length of 20 baseswas designed comprising in the 5′ to 3′ direction: a 5× triplet repeatwhich is different from that on the second oligonucleotide probe; aneutral spacer comprising 5× Thymidine bases and a 3′ Biotin molecule,added during synthesis. The control oligonucleotide binds to its reversecomplement on the lateral flow strip to verify a successful carbonlateral flow procedure.

Reactions were prepared containing: 1.8 pmol of the first primer; 9.6pmol of the second primer; 3.6 pmol of the first probe; 1 pmol of thesecond probe; 300 μM Sp-dATP-α-S from Enzo Life Sciences; 60 μM of eachof dTTP, dCTP and dGTP; 28U of the restriction enzyme; 14U of a Bacillusstrand displacement DNA polymerase; 35U of a viral reverse transcriptaseenzyme; 3.5U RNaseH and 3 μg carbon adsorbed to biotin binding protein.5 μl of nasopharyngeal swab sample collected from patients in a clinicalsetting (sourced from Discovery Life Sciences) which included 7 viruspositive samples and 6 virus negative clinical samples (verified by PCRassay). Reactions were performed in a 70 μl volume in an appropriatereaction buffer. Reactions were incubated at 45° C. for 4 min 30 secbefore the entire reaction was loaded onto a nucleic acid lateral flowstrip printed with approximately 50 pmol of the reverse complement tothe 3 base triplet repeat moiety of the second oligonucleotide probe(bottom) and the reverse complement to the control oligonucleotide (topline).

FIG. 11 displays a photograph of the lateral flow strips obtained in theperformance of the example. The arrows indicate the position where thereverse complement to the triplet repeat moiety of the secondoligonucleotide probe has been printed (+) and hence where the positivesignal appears, and the position of the reverse complement to thecontrol oligonucleotide (CTL) which verifies a successful lateral flowrun and hence appears in both positive and negative assays. The toppanel (+ve) shows the results obtained with the virus positive clinicalsamples and the bottom panel (−ve) those with the virus negativesamples. A clear black line indicating the presence of target nucleicacid is present in each of the positive samples, demonstrating the rapiddetection of clinical specimens by the method of the invention. No falsepositives were observed, demonstrating the complete absence ofnon-specific production of the detector species, such as through abinitio synthesis or primer-primer binding. No false negatives wereobserved evidencing the robustness of the method and its sensitivityacross the different target nucleic acid copy number levels presentwithin different clinical specimens.

Example 8 Performance of the Method at Different Temperatures

The method of the invention may be performed efficiently over a widerange of temperatures and does not require temperature cycling, nor anyhot or warm start, pre-heating or a controlled temperature decrease.This example demonstrates the performance of a typical assay over arange of different temperatures. By selecting enzymes with the desiredtemperature optima, and using a phosphorothioate base that reduces themelting temperature of hybridisation following its incorporation, asassay has been readily developed wherein the amplification is performedover a surprisingly wide range of temperatures and covering an usuallylow temperature range. A separate experiment further demonstrates thatassays developed using the method of the invention can be developed withno requirement to preheat the sample prior to the initiation of step a),and wherein no loss of performance is observed when the temperature isincreased during the performance of the amplification in step a).

Example 8.1: An assay was designed exploiting the embodiment of themethod wherein the first oligonucleotide probe is blocked at the 3′ endfrom extension by the DNA polymerase and is not capable of being cleavedby either the first or second restriction enzyme and is contacted withthe sample simultaneously to the performance of step a). A first primerwas designed containing in the 5′ to 3′ direction: a neutral region of 7bases; the recognition site of a restriction enzyme; and, a 11 baseregion capable of hybridising to the first hybridisation sequence in thetarget nucleic acid, a DNA target. A second primer was designedcontaining in the 5′ to 3′ direction: a neutral region of 7 bases; therecognition site for the same restriction enzyme as the first primer;and a 12 base region capable of hybridising to the reverse complement ofthe second hybridisation sequence in the target nucleic acid.

A first oligonucleotide probe was designed with a total length of 21bases comprising in the 5′ to 3′ direction: a 5′ Biotin modification; aneutral region of 6 bases; the bases of the recognition site of therestriction enzyme containing a mismatch at the 2 position; a 10 baseregion capable of hybridising to the first hybridisation region in thetarget comprising a G-clamp modification at the 6^(th) position; and a3′ phosphate modification, wherein the biotin modification permitsattachment of the first oligonucleotide probe to a colorimetric dye,carbon nanoparticles, and the phosphate modification blocks itsextension by the strand displacement DNA polymerase.

A second oligonucleotide probe was designed containing in the 5′ to 3′direction: an 11 base region capable of hybridising to the reversecomplement of the second hybridisation sequence in the target; a 4×Thymidine base spacer and 12 bases comprising 4× repeats of a 3 basesequence motif which acts as the moiety permitting the attachment of thesecond oligonucleotide probe to a solid material. An additional singlestranded oligonucleotide was designed comprising in the 5′ to 3′direction: an 11× Thymidine base spacer; a 33 base region comprising a11× repeat of the reverse complement to the 3 base sequence motif whichforms the moiety permitting attachment of the second oligonucleotide toa solid material. For the second oligonucleotide probe nucleic acidlateral flow strips were prepared spotted with 30 pmol of saidadditional single stranded oligonucleotide.

Reactions were prepared in appropriate buffer containing: 1.5 pmol ofthe first primer; 1.0 pmol of the second primer; 1 pmol of the firstoligonucleotide probe; 60 μM Sp-dATP-α-S from Enzo Life Sciences; 60 μMof each of dTTP, dCTP and dGTP; and, various levels of target DNA (++=1amol, +=10 zmol, NTC=no target control). Assembled reactions wereincubated for 2 min at the target temperature (I=37° C.; II=45° C.,III=50° C. and IV=55° C.) before being initiated by final addition of 5Uof the restriction enzyme and 5U of a Bacillus strand displacement DNApolymerase to a final reaction volume of 25 μl. Reactions were thenincubated for 5 min (T1) or 8 min (T2) at the relevant targettemperature. Following incubation, each reaction was transferred to 75μl of buffer containing 1.5 pmol of the second oligonucleotide probe and8 μg of carbon adsorbed to biotin binding protein before application tothe sample pad of the nucleic acid lateral flow strip.

FIG. 12A displays photographs of the lateral flow strips obtained in theexperiment at each target level, temperature and timepoint. The clearblack lines observed correspond to the deposition of carbon attacheddetector species produced in the presence of target. At all temperaturesa very strong signal appeared in the presence of target at both targetlevels within 8 min demonstrating the broad temperature range ofefficient amplification of the method. No non-specific amplification wasobserved in the NTC samples. Strong amplification was also observedafter just 5 min at 45° C. and 50° C. indicating that the optimumtemperature for this assay is likely to be between 40° C. and 50° C.

Example 8.2: A second assay was designed exploiting the embodiment ofthe method wherein the first oligonucleotide probe is blocked at the 3′end from extension by the DNA polymerase and is not capable of beingcleaved by either the first or second restriction enzyme and contactedwith the sample simultaneously to the performance of step a). Both thefirst and second primers were designed to contain in the 5′ to 3′direction: a neutral region of 6 bases; the recognition site of arestriction enzyme; and a 12 base hybridisation region for the targetnucleic acid. The primers were designed such that the first and secondhybridisation sequences in the target are separated by 10 bases.

A first oligonucleotide probe was designed with a total length of 23bases comprising in the 5′ to 3′ direction: a 5′ Biotin modification; aneutral region of 6 bases; the bases of the recognition site of therestriction enzyme containing a mismatch at the 4th position; a 12 baseregion capable of hybridising to the first hybridisation region in thetarget; and a 3′ phosphate modification, wherein the biotin modificationpermits attachment of the first oligonucleotide probe to a colorimetricdye, carbon nanoparticles, and the phosphate modification blocks itsextension by the strand displacement DNA polymerase.

A second oligonucleotide probe was designed containing in the 5′ to 3′direction: a 13 base region capable of hybridising to 3 bases of thereverse complement of the second hybridisation sequence in the targetand the 10 base gap between the first and second hybridisationsequences; a 3× Thymidine base spacer and 12 bases comprising 4× repeatsof a 3 base sequence motif which acts as the moiety permitting theattachment of the second oligonucleotide probe to a solid material. Anadditional single stranded oligonucleotide was designed comprising inthe 5′ to 3′ direction: an 11× Thymidine base spacer; and a 36 baseregion comprising a 12× repeat of the reverse complement to the 3 basesequence motif which forms the moiety permitting attachment of thesecond oligonucleotide to a solid material. For the secondoligonucleotide probe nucleic acid lateral flow strips were preparedspotted with 30 pmol of said additional single stranded oligonucleotide.

Reactions were prepared in appropriate buffer containing: 6 pmol of thefirst oligonucleotide primer; 8 pmol of the second oligonucleotideprimer; 6 pmol of the first oligonucleotide probe; 60 μM Sp-dATP-α-Sfrom Enzo Life Sciences; 60 μM of each of dTTP, dCTP and dGTP; 60 μg ofcarbon adsorbed to biotin binding protein; and, where applicable,target. Assembled reactions were incubated for 2 min at the startingtemperature (I=15° C.; II=45° C.) before reactions were initiated byfinal addition of 20U of the restriction enzyme, 20U of a Bacillusstrand displacement DNA polymerase and 40U of reverse transcriptase to afinal reaction volume of 100μ1. Following enzyme addition, the reactionswith the 15° C. starting temperature were immediately transferred to 45°C. alongside the other reactions.

Reactions were then incubated for 6 min at 45° C. Following incubation,each reaction was transferred to the sample pad of a nucleic acidlateral flow strip, which sample pad contained 3 pmol of the secondoligonucleotide probe. FIG. 12B displays photographs of the lateral flowstrips obtained in the experiment at each temperature incubationconditions. The clear black lines observed correspond to the depositionof carbon attached detector species produced in the presence of target.No difference was observed in the reaction wherein the temperature hadbeen increased from 15° C. to 45° C. during the amplification step a).The same remarkable rate of amplification occurred as in the pre-heatedreaction, and no non-specific amplification was observed in the NTCsample.

This Example 8 demonstrates that the method of the invention can be usedto readily develop assays with a lower optimal temperature profilecompared to known methods, and which can be exploited for sensitivedetection over an unusually broad range of temperatures. It alsodemonstrates that the method of the invention can be performed withoutpreheating wherein the temperature is increased during the performanceof step a). Such features are highly attractive for use of the method ina low-cost diagnostic device, where high temperatures and preciselycontrolled heating impose complex physical constraints that increase thecost-of-goods of such a device to a point where a single-use orinstrument-free device is not commercially viable. Furthermore byavoiding the requirement of known methods to pre-heat the sample priorto the initiation of amplification, the method of the invention can beperformed with fewer user steps and a simpler sequence of operations,thus increasing the usability of such a diagnostic device and decreasingthe overall time to result.

Example 9 PERFORMANCE of the Method Wherein the Target Nucleic Acid isDerived from Double Stranded DNA by Strand Invasion

This example demonstrates the use of the method wherein the singlestranded target nucleic acid is a single stranded site within doublestranded DNA that is detected without any requirement for specificaction to separate the DNA strands, such as temperature denaturation,bump primers or use of an additional enzyme (e.g. helicase orrecombinase). The ability to use the method of the invention readily forthe detection of both single-stranded RNA and double-stranded DNAtargets makes it highly versatile for use in diagnostic applications,without additional user steps, components or physical requirementsimposed on the device used to perform the method.

Example 9.1: An assay was developed for a protein coding region withinthe double-stranded DNA genome a viral target. It is possible to useeither the double-stranded genome or the mRNA transcript as a biomarkerfor the presence of the virus in clinical diagnosis. The assay wasdesigned exploiting the embodiment of the method wherein the firstoligonucleotide probe is blocked at the 3′ end from extension by the DNApolymerase and is not capable of being cleaved by either the first orsecond restriction enzyme and is contacted with the samplesimultaneously to the performance of step a). The design of theoligonucleotide primers and oligonucleotide probes was performedfollowing a similar approach to that described in other examples, withno gap between the first and the second hybridisation sequences in thetarget nucleic acid.

Reactions were prepared in appropriate buffer containing: 4 pmol of thefirst oligonucleotide primer; 2 pmol of the second oligonucleotideprimer; 2 pmol of the first oligonucleotide probe; 60 μM Sp-dATP-α-Sfrom Enzo Life Sciences; 60 μM of each of dTTP, dCTP and dGTP; 60 μg ofcarbon adsorbed to biotin binding protein; and either double strandedDNA target (I) or single-stranded RNA target (II) or no target.Assembled reactions were incubated for 2 min at the 45° C. before beinginitiated by final addition of 20U of the restriction enzyme, 20U of aBacillus strand displacement DNA polymerase and 25U of reversetranscriptase to a final reaction volume of 100μ1. Following enzymeaddition, the reactions were incubated at 45° C. for 7 min.

Following incubation, 1.5 pmol of the second oligonucleotide probe wasadded to each reaction and the entire reaction volume was transferred tothe sample pad of a nucleic acid lateral flow strip. A nucleic acidlateral flow control target was also added to all samples. FIG. 13Adisplays photographs of the lateral flow strips obtained in theexperiment with each target. The clear black lines observed correspondto the deposition of carbon attached detector species produced in thepresence of target, with a fainter signal corresponding to the lowertarget level (+) than the higher target lever (++). No difference in therate of amplification was observed between the single stranded RNA anddouble stranded DNA targets.

Example 9.2: An assay was designed for a single stranded target nucleicacid within the c.2.5 megabase double stranded DNA genome of a bacterialpathogen. The assay was designed exploiting the embodiment of the methodwherein the first oligonucleotide probe is blocked at the 3′ end fromextension by the DNA polymerase and is not capable of being cleaved byeither the first or second restriction enzyme and is contacted with thesample simultaneously to the performance of step a). The design of theoligonucleotide primers and oligonucleotide probes was performedfollowing a similar approach to that described in other examples, with agap of 4 bases between the first and the second hybridisation sequencesin the target nucleic acid.

Reactions were prepared in appropriate buffer containing 2 pmol of thefirst oligonucleotide primer and 0.5 pmol of the second oligonucleotideprimer. Due to the use of a double stranded DNA target, two singlestranded target nucleic acids are in fact added at the same time and itis assumed that a second reciprocal process also occurs, wherein thesecond oligonucleotide primer for detection of the target nucleic acidis the first oligonucleotide primer for the detection of the secondtarget nucleic acid, being the reverse complement of the target nucleicacid. This fact has little impact on the performance of the method. 2pmol of the first oligonucleotide probe; 60 μM Sp-dATP-α-S from EnzoLife Sciences; 60 μM of each of dTTP, dCTP and dGTP; 15 μg of carbonadsorbed to biotin binding protein; and genome extract of the bacteriacontaining the target at various levels (++=1 amol; +=10 zmol; NTC=notarget control). A further specificity control reaction was alsoperformed containing 1 amol of genome extract of E. coli. Assembledreactions were incubated for 3 min at 45° C. before being initiated byfinal addition of 4U of the restriction enzyme and 2U of a Bacillusstrand displacement DNA polymerase to a final reaction volume of 25 μl.Following enzyme addition, the reactions were incubated at 45° C. for 6min.

Following incubation, 75 μl buffer containing 3 pmol of the secondoligonucleotide probe was added to each reaction and the entire volumewas then transferred to the sample pad of a nucleic acid lateral flowstrip. FIG. 13B displays photographs of the lateral flow strips obtainedin the experiment with each target. Clear black lines corresponding tothe deposition of carbon attached detector species produced in thepresence of target are observed at both target levels tested. Nonon-specific signal was observed in the no target control or in thepresence of E. coli genomic DNA demonstrating that the method can beemployed for the specific detection of a complex double-stranded DNAgenome at a clinically relevant copy number within just 6 min.

This example demonstrates that the method of the invention can bereadily used for the detection of single stranded nucleic acid targetswithin double stranded DNA. Remarkably, a similar rate of amplificationis observed for the detection of single stranded RNA target and the sametarget sequence within double stranded DNA, without any requirement forspecific action such as temperature denaturation to separate the DNAduplex. Instead the single stranded site is exposed sufficiently forhybridisation and extension of the first oligonucleotide primer toinitiate the method by “strand invasion” wherein transient opening ofone or more DNA base pairs within the double stranded DNA occurssufficiently to permit hybridisation and extension of the 3′ hydroxyl ofthe first oligonucleotide primer. This contrasts with known methods suchas SDA wherein heat denaturation and bump primers are utilised in assaysfor double stranded nucleic acids. The ability to use the method of theinvention readily to detect targets within double-stranded DNA inaddition to those within single stranded DNA and single stranded RNAmakes it highly versatile for use in diagnostic applications, such as inthe detection of bacterial, fungal and viral pathogens that have adouble stranded DNA genome. The fact that the method has no requirementfor complex additional user steps, enzymes, components or physicalconstraints to detect organisms with double stranded DNA genomes meansit is particularly well-suited for testing in a simple, low-costdiagnostic device. For example, the requirement for heat denaturationprior to performance of amplification reported for known methods wouldnecessitate expensive additional components and increase the costs ofgoods of such a device and the total time to result, meaning that asingle-use or self-contained, instrument free device would not beviable.

Example 10 Comparative Performance of the Method of the Invention VersusKnown Methods

This example presents a comparative evaluation of the method of theinvention against the known method disclosed in WO2014/164479 for thedetection of a viral target. The known method is fundamentally differentto the method of the invention in that it requires nicking enzymes anddoes not require the use of one or more modified dNTP. The method of theinvention is demonstrated to have vastly superior sensitivity andspecificity.

For this comparative evaluation an assay was first developed for a viraltarget with a single-stranded RNA genome using the method of theinvention. Said assay was designed exploiting the embodiment of themethod wherein the first oligonucleotide probe is blocked at the 3′ endfrom extension by the DNA polymerase and is not capable of being cleavedby either the first or second restriction enzyme and contacted with thesample simultaneously to the performance of step a). The design of theoligonucleotide primers and oligonucleotide probes was performedfollowing a similar approach to that described in other examples, with agap of 6 bases between the first and second hybridisation sequences inthe target nucleic acid.

For the assay using the known method, similar primers were designedcontaining the same 6 base neutral region at the 5′ ends and the samehybridisation regions at the 3′ end as the equivalent primers used inthe method of the invention. In this way consistency was ensured as muchas possible between the two assays for an accurate comparison of themethods. However, the bases of the restriction enzyme recognition sitewere replaced with those of the exemplary nicking enzyme reported inWO2014/164479, Nt.BbvCI (see Example 5 on p. 20-21).

Example 10.1: In the first instance, reactions for each method wereperformed using equal primer ratios. For the method of the invention,reactions were prepared in appropriate buffer containing: 2 pmol of thefirst oligonucleotide primer; 2 pmol of the second oligonucleotideprimer; 1.6 pmol of the first oligonucleotide probe; 60 μM Sp-dATP-α-Sfrom Enzo Life Sciences; 60 μM of each of dTTP, dCTP and dGTP; and viralgenomic RNA extract at various levels as target (+++=10 zmol; ++=100copies; +=10 copies; NTC=no target control). Assembled reactions werepreincubated for 5 min at ambient conditions (c.20° C.) before reactionswere initiated by addition of 5U of the restriction enzyme, 5U of aBacillus strand displacement DNA polymerase and 10U of reversetranscriptase in a final reaction volume of 25 μl. Following enzymeaddition, the reactions were incubated at 45° C. for 8 min (T1) or 15min (T2). Following incubation, 60 μg of carbon adsorbed to biotinbinding protein in 75 μl buffer was added to each reaction and theentire 100 μl volume was transferred to a nucleic acid lateral flowstrip containing 1.5 pmol of the second oligonucleotide probe on thesample pad.

For the known method, reactions were prepared in appropriate buffercontaining: 6.25 pmol of the first oligonucleotide primer; 6.25 pmol ofthe second oligonucleotide primer; 200 μM of each of dATP, dTTP, dCTPand dGTP; and viral genomic RNA extract at various levels as target(+++=10 zmol; ++=100 copies; +=10 copies; NTC=no target control).Assembled reactions were preincubated for 5 min at ambient conditions(c.20° C.) before reactions were initiated by addition of 4U ofNt.BbvCI, 20U of Bst large fragment DNA polymerase and 10U of M-MuLVreverse transcriptase in a final reaction volume of 25 μl. Followingenzyme addition, the reactions were incubated at 45° C. for 8 min (T1)and 15 min (T2). Following incubation, 75 μl buffer containing 60 μgcarbon adsorbed to biotin binding protein and 5 pmol of the firstoligonucleotide probe was added to each reaction and the entire 100 μlvolume was transferred to a nucleic acid lateral flow strip containing 5pmol of the second oligonucleotide probe on the sample pad.

FIG. 14A displays photographs of the lateral flow strips obtained in theexperiment with the method of the invention (I) and with the knownmethod (II), at the various target levels and time points indicated. Theblack lines observed correspond to the deposition of carbon attacheddetector species produced in the presence of target. Several attemptswere required before it was possible to observe any signal at all usingthe known method and it was necessary to use a particular combination ofenzymes and buffer and significantly higher levels of primers, dNTPs andenzymes. With the method of the invention (I), even at the shortest timepoint after just 8 min without a pre-heat it was possible to clearly seethe detector species produced even at the lowest target level of just 10copies of target. Even after efforts to optimise the known method whichwould not have been obvious to the skilled person, only a faint signalwas observed at the highest target level (+++=10 zmol) and at thelongest time point (15 min).

Example 10.2: After extensive further, non-obvious, attempts it waspossible to increase the performance of the known method, but only byusing a 2:1 ratio of the first and second oligonucleotide primers, witha very high concentration of the first primer, as described in thisExample 10.2. The method of the invention was performed again asdescribed in Example 10.1. For the known method, the reactions wereperformed as described in Example 10.1 except that the level of thefirst oligonucleotide primer was increased to 12.5 pmol. In each casethe following target levels were used: +++=1 zmol; ++=100 copies; +=10copies; NTC=no target control.

FIG. 14B displays photographs of the lateral flow strips obtained in theexperiment with the method of the invention (I) and with the knownmethod (II), at the various target levels and time points indicated. Theblack lines observed correspond to the deposition of carbon attacheddetector species produced in the presence of target. Again, the methodof the invention (I) demonstrated a remarkable rate with signal visibleeven at the shortest time point and at the lowest target level of just10 copies of target. With the known method only a faint signal wasobserved at the highest target level (+++=1 zmol) and a very faintsignal was visible in the 100 copy sample at the longest time point (15min). However, a faint signal was also observed in the NTC strip whichmay correspond to non-specific product as a result of the very higholigonucleotide primer levels and enzyme levels required to get themethod to work at all. These data are consistent with the data inWO2014/164479 wherein an incubation time of 30 min was reported. Therequirement to add unusually high primer levels in order to speed up theamplification performed using this known method would greatly limit itspotential application to the detection of two or more different targetsin the same sample, as there would be very limited scope to furtherincrease the total primer level without exacerbating the problem withnon-specific products.

This Example 14 demonstrates the striking superiority of the method ofthe invention over the known method disclosed in WO2014/164479 withamplification performed much more rapidly, with greater sensitivity andwith a more clear results signal produced. In just 8 min withoutpre-incubation the method of the invention produced a stronger signalwith just 100 copies of target than the known method was able to in 15min at the highest target level with 60× the level of target. Theadvantages of the method of the invention over this known method arisefrom its requirement for a different class of enzyme, being restrictionenzymes that are not nicking enzymes, and from its requirement for useof one or more modified dNTP, such as a phosphorothioate base whichenhances the sensitivity and specificity of amplification. Furthermore,the embodiment of the method wherein one of the first and secondoligonucleotide probes is blocked at the 3′ end from extension by theDNA polymerase and is not capable of being cleaved by either the firstor second restriction enzyme and is contacted with the samplesimultaneously to the performance of step a), enables efficient couplingof amplification to signal detection and facilitates enhancedspecificity derived from efficient sequence based hybridisation duringthe formation of the detector species. These advantages make the methodof the invention ideally suited to exploitation in the field ofdiagnostics and to the development of simple, ultra-rapid, user-centred,low-cost diagnostic devices, such as a single-use or instrument freemolecular diagnostic test device. Throughout the specification and theclaims which follow, unless the context requires otherwise, the word‘comprise’, and variations such as ‘comprises’ and ‘comprising’, will beunderstood to imply the inclusion of a stated integer, step, group ofintegers or group of steps but not to the exclusion of any otherinteger, step, group of integers or group of steps.

Additional aspects of the invention include those listed below:

1. A method for detecting the presence of a single stranded targetnucleic acid of defined sequence in a sample comprising:

-   -   a) contacting the sample with:        -   i. a first oligonucleotide primer and a second            oligonucleotide primer wherein said first primer comprises            in the 5′ to 3′ direction one strand of a restriction enzyme            recognition sequence and cleavage site and a region that is            capable of hybridising to a first hybridisation sequence in            the target nucleic acid, and said second primer comprises in            the 5′ to 3′ direction one strand of a restriction enzyme            recognition sequence and cleavage site and a region that is            capable of hybridising to the reverse complement of a second            hybridisation sequence upstream of the first hybridisation            sequence in the target nucleic acid;        -   ii. a strand displacement DNA polymerase;        -   iii. dNTPs;        -   iv. one or more modified dNTP;        -   v. a first restriction enzyme that is not a nicking enzyme            but is capable of recognising the recognition sequence of            the first primer and cleaving only the first primer strand            of the cleavage site when said recognition sequence and            cleavage site are double stranded, the cleavage of the            reverse complementary strand being blocked due to the            presence of one or more modifications incorporated into said            reverse complementary strand by the DNA polymerase using the            one or more modified dNTP; and        -   vi. a second restriction enzyme that is not a nicking enzyme            but is capable of recognising the recognition sequence of            the second primer and cleaving only the second primer strand            of the cleavage site when said recognition sequence and            cleavage site are double stranded, the cleavage of the            reverse complementary strand being blocked due to the            presence of one or more modifications incorporated into said            reverse complementary strand by the DNA polymerase using the            one or more modified dNTP;        -   to produce, without temperature cycling, in the presence of            said target nucleic acid, amplification product;    -   b) contacting the amplification product of step a) with:        -   i. a first oligonucleotide probe which is capable of            hybridising to a first single stranded detection sequence in            at least one species within the amplification product and            which is attached to a moiety that permits its detection;            and        -   ii. a second oligonucleotide probe which is capable of            hybridising to a second single stranded detection sequence            upstream or downstream of the first single stranded            detection sequence in said at least one species within the            amplification product and which is attached to a solid            material or to a moiety that permits its attachment to a            solid material;        -   where hybridisation of the first and second probes to said            at least one species within the amplification product            produces a detector species; and    -   c) detecting the presence of the detector species produced in        step b) wherein the presence of the detector species indicates        the presence of the target nucleic acid in said sample.        2. A method according to aspect 1 wherein one of the first and        second oligonucleotide probes is blocked at the 3′ end from        extension by the DNA polymerase and is not capable of being        cleaved by either the first or second restriction enzymes.        3. A method according to aspect 2 wherein the one        oligonucleotide probe is rendered not capable of being cleaved        by either the first or second restriction enzymes due to the        presence of one or more sequence mismatch and/or one or more        modifications such as a phosphorothioate linkage.        4. A method according to aspect 2 or 3 wherein the one        oligonucleotide probe is contacted with the sample        simultaneously to the performance of step a).        5. A method according to any of the preceding aspects wherein        the sample additionally is contacted in step a) with: (A) a        third oligonucleotide primer which third primer comprises in the        5′ to 3′ direction one strand of the recognition sequence and        cleavage site for the first restriction enzyme and a region that        is capable of hybridising to the first hybridisation sequence in        the target nucleic acid and wherein said third primer is blocked        at the 3′ end from extension by the DNA polymerase; and/or (B) a        fourth oligonucleotide primer which fourth primer comprises in        the 5′ to 3′ direction one strand of the recognition sequence        and cleavage site for the second restriction enzyme and a region        that is capable of hybridising to the reverse complement of the        second hybridisation sequence in the target sequence and wherein        said fourth primer is blocked at the 3′ end from extension by        the DNA polymerase.        6. A method according to aspect 5 wherein when present the third        oligonucleotide primer is provided in excess of the first        oligonucleotide primer and when present the fourth        oligonucleotide primer is provided in excess of the second        oligonucleotide primer.        7. A method according to any of the preceding aspects wherein        the one or more modified dNTP is an alpha thiol modified dNTP.        8. A method according to any of the preceding aspects wherein        the first and second restriction enzyme are the same restriction        enzyme.        9. A method according to any of the preceding aspects wherein        two or more of steps a), b) and c) are performed simultaneously.        10. A method according to any of the preceding aspects wherein        step (a) is performed at a temperature of not more than 50° C.        11. A method according to any of the preceding aspects wherein        the moiety that permits the detection of the first        oligonucleotide probe, is a colorimetric or fluorometric dye or        a moiety that is capable of attachment to a colorimetric or        fluorometric dye such as biotin.        12. A method according to any of the preceding aspects wherein        the detector species is detected by a change in electrical        signal.        13. A method according to any of the preceding aspects wherein        the moiety that permits the detection of the first        oligonucleotide probe is an enzyme that yields a detectable        signal, such as a colorimetric or fluorometric signal, following        contact with a substrate.        14. A method according to any of the preceding aspects wherein        the moiety that permits the attachment of the second        oligonucleotide probe to a solid material is a single stranded        oligonucleotide.        15. A method according to aspect 14 wherein the sequence of the        single stranded oligonucleotide moiety comprises three or more        repeat copies of a 2 to 4 base DNA sequence motif.        16. A method according to any of the preceding aspects wherein        in step c) the presence of the detector species is detected by        nucleic acid lateral flow.        17. A method according to aspect 16 wherein the nucleic acid        lateral flow utilises one or more nucleic acids that is capable        of sequence specific hybridisation to the moiety that permits        the attachment of the second oligonucleotide probe to a solid        material.        18. A method according to any of the preceding aspects wherein        step c) produces a colorimetric or electrochemical signal using        carbon or gold, preferably carbon.        19. A method according to any of the preceding aspects wherein        the first and/or second oligonucleotide primers comprise a        stabilising sequence at the 5′ end, e.g. of 5 bases in length        upstream of the restriction enzyme recognition sequence and        cleavage site.        20. A method according to any of the preceding aspects wherein        the hybridising region of the first and/or second        oligonucleotide primers is between 9 and 16 bases in length.        21. A method according to any of the preceding aspects wherein        one of the first and second oligonucleotide primers is provided        in excess of the other.        22. A method according to any of the preceding aspects wherein        the first and second hybridisation sequences in the target        nucleic acid are separated by 0 to 6 bases.        23. A method according to any of the preceding aspects wherein        the first and second hybridisation sequences in the target        nucleic acid are separated by 3 to 6 bases.        24. A method according to any of the preceding aspects wherein        in step b) either the first or second single stranded detection        sequence in the at least one species within the amplification        product includes the sequence corresponding to the 3 to 6 bases        defined in claim 23.        25. A method according to any of the preceding aspects wherein        the level of the target nucleic acid in said sample is        quantified in step c).        26. A method according to any of the preceding aspects wherein        the target nucleic acid is single stranded RNA, including single        stranded RNA derived from double stranded RNA and single        stranded RNA derived from double stranded DNA, or single        stranded DNA, including single stranded DNA derived from single        stranded RNA and single stranded DNA derived from double        stranded DNA.        27. A method according to aspect 26 wherein said single stranded        DNA is derived from double stranded DNA by use of a nuclease,        such as a restriction endonuclease or exonuclease III or derived        from single stranded RNA by use of reverse transcriptase.        28. A method according to any of the preceding aspects wherein        the presence of two or more different target nucleic acids of        defined sequence are detected in the same sample.        29. A method according to any of the preceding aspects wherein        the sample is a biological sample, such as a nasal or        nasopharyngeal swab or aspirate, blood or a sample derived from        blood, or urine.        30. A method according to any of the preceding aspects wherein        the target nucleic acid is viral or derived from viral nucleic        acid material, is bacterial or derived from bacterial nucleic        acid material, is circulating, cell-free DNA released from        cancer cells or foetal cells, is micro RNA or derived from micro        RNA.        31. A method according to any of the preceding aspects wherein        the target nucleic acid contains a site of epigenetic        modification, such as methylation.        32. A method according to any of the preceding aspects wherein        the detection of the target nucleic acid is used for the        diagnosis, prognosis or monitoring of a disease or a diseased        state.        33. A method according to aspect 32 wherein said disease is an        infectious disease, including but not limited to HIV, influenza,        RSV, Rhinovirus, norovirus, tuberculosis, HPV, meningitis,        hepatitis, MRSA, Ebola, Clostridium difficile, Epstein-Barr        virus, malaria, plague, polio, chlamydia, herpes, gonorrhoea,        measles, mumps, rubella, cholera or smallpox.        34. A method according to aspect 32 wherein said disease is a        cancer, including but not limited to colorectal cancer, lung        cancer, breast cancer, pancreatic cancer, prostate cancer, liver        cancer, bladder cancer, leukaemia, esophageal cancer, ovarian        cancer, kidney cancer, stomach cancer or melanoma.        35. A method according to any of the preceding aspects wherein        the detection of said target nucleic acid is used for human        genetic testing, prenatal testing, blood contamination        screening, pharmacogenomics or pharmacokinetics.        36. A method according to any of the preceding aspects wherein        the sample is a human sample, a forensic sample, an agricultural        sample, a veterinary sample, an environmental sample or a        biodefence sample.        37. A kit comprising:    -   a) a first oligonucleotide primer and a second oligonucleotide        primer wherein said first primer comprises in the 5′ to 3′        direction a restriction enzyme recognition sequence and cleavage        site and a region that is capable of hybridising to a first        hybridisation sequence in a single stranded target nucleic acid        of defined sequence, and said second primer comprises in the 5′        to 3′ direction a restriction enzyme recognition sequence and        cleavage site and a region that is capable of hybridising to the        reverse complement of a second hybridisation sequence upstream        of the first hybridisation sequence in the target nucleic acid;    -   b) a first restriction enzyme that is not a nicking enzyme and        is capable of recognising the recognition sequence of and        cleaving the cleavage site of the first primer and a second        restriction enzyme that is not a nicking enzyme and is capable        of recognising the recognition sequence of and cleaving the        cleavage site of the second primer;    -   c) a strand displacement DNA polymerase;    -   d) dNTPs;    -   e) one or more modified dNTP;    -   f) a first oligonucleotide probe which has some complementarity        to the hybridising region of one of the first and second        oligonucleotide primers and is attached to a moiety that permits        its detection; and    -   g) a second oligonucleotide probe which has some complementarity        to the reverse complement of the hybridising region of the other        of the first and second oligonucleotide primer and is attached        to a solid material or to a moiety that permits its attachment        to a solid material.        38. A kit according to aspect 37 which additionally comprises        means to detect the presence of the detector species.        39. A kit according to aspect 37 or 38 wherein the first        oligonucleotide primer and/or the second oligonucleotide primer        and/or the first restriction enzyme and/or the second        restriction enzyme and/or the DNA polymerase and/or the dNTPs        and/or the one or more modified dNTP and/or the first        oligonucleotide probe and/or the second oligonucleotide probe        are as defined in any one of aspects 2, 3, 7, 8, 11, 13 to 17,        19, 20 or 22 to 24.        40. A kit according to any of aspects 37 to 39 which        additionally comprises third and/or fourth oligonucleotide        primers as defined in aspect 5 or 6.        41. A method for detecting the presence of a single stranded        target nucleic acid of defined sequence in a sample comprising:    -   a) contacting the sample with:        -   i. a first oligonucleotide primer and a second            oligonucleotide primer wherein said first primer comprises            in the 5′ to 3′ direction one strand of a restriction enzyme            recognition sequence and cleavage site and a region that is            capable of hybridising to a first hybridisation sequence in            the target nucleic acid, and said second primer comprises in            the 5′ to 3′ direction one strand of a restriction enzyme            recognition sequence and cleavage site and a region that is            capable of hybridising to the reverse complement of a second            hybridisation sequence upstream of the first hybridisation            sequence in the target nucleic acid;        -   ii. a strand displacement DNA polymerase;        -   iii. dNTPs;        -   iv. one or more modified dNTP;        -   v. a first restriction enzyme that is not a nicking enzyme            but is capable of recognising the recognition sequence of            the first primer and cleaving only the first primer strand            of the cleavage site when said recognition sequence and            cleavage site are double stranded, the cleavage of the            reverse complementary strand being blocked due to the            presence of one or more modifications incorporated into said            reverse complementary strand by the DNA polymerase using the            one or more modified dNTP; and        -   vi. a second restriction enzyme that is not a nicking enzyme            but is capable of recognising the recognition sequence of            the second primer and cleaving only the second primer strand            of the cleavage site when said recognition sequence and            cleavage site are double stranded, the cleavage of the            reverse complementary strand being blocked due to the            presence of one or more modifications incorporated into said            reverse complementary strand by the DNA polymerase using the            one or more modified dNTP;        -   to produce, without temperature cycling, in the presence of            said target nucleic acid, amplification product;    -   b) contacting the amplification product of step a) with:        -   i. a first oligonucleotide probe which is capable of            hybridising to a first single stranded detection sequence in            at least one species within the amplification product and            which is attached to a moiety that permits its detection;            and        -   ii. a second oligonucleotide probe which is capable of            hybridising to a second single stranded detection sequence            upstream or downstream of the first single stranded            detection sequence in said at least one species within the            amplification product and which is attached to a solid            material or to a moiety that permits its attachment to a            solid material;        -   wherein one of the first and second oligonucleotide probes            is blocked at the 3′ end from extension by the DNA            polymerase and is not capable of being cleaved by either the            first or second restriction enzymes, and where hybridisation            of the first and second probes to said at least one species            within the amplification product produces a detector            species; and    -   c) detecting the presence of the detector species produced in        step b) wherein the presence of the detector species indicates        the presence of the target nucleic acid in said sample;    -   and wherein either the first or second oligonucleotide probe        defined in step b) is contacted with the sample simultaneously        to the performance of step a).

All patents and patent applications referred to herein are incorporatedby reference in their entirety.

1. A method for detecting the presence of a single stranded targetnucleic acid of defined sequence in a sample comprising: a) contactingthe sample with: i. a first oligonucleotide primer and a secondoligonucleotide primer wherein said first primer comprises in the 5′ to3′ direction one strand of a restriction enzyme recognition sequence andcleavage site and a region that is capable of hybridising to a firsthybridisation sequence in the target nucleic acid, and said secondprimer comprises in the 5′ to 3′ direction one strand of a restrictionenzyme recognition sequence and cleavage site and a region that iscapable of hybridising to the reverse complement of a secondhybridisation sequence upstream of the first hybridisation sequence inthe target nucleic acid; ii. a strand displacement DNA polymerase; iii.dNTPs; iv. one or more modified dNTP; v. a first restriction enzyme thatis not a nicking enzyme but is capable of recognising the recognitionsequence of the first primer and cleaving only the first primer strandof the cleavage site when said recognition sequence and cleavage siteare double stranded, the cleavage of the reverse complementary strandbeing blocked due to the presence of one or more modificationsincorporated into said reverse complementary strand by the DNApolymerase using the one or more modified dNTP; and vi. a secondrestriction enzyme that is not a nicking enzyme but is capable ofrecognising the recognition sequence of the second primer and cleavingonly the second primer strand of the cleavage site when said recognitionsequence and cleavage site are double stranded, the cleavage of thereverse complementary strand being blocked due to the presence of one ormore modifications incorporated into said reverse complementary strandby the DNA polymerase using the one or more modified dNTP; to produce,without temperature cycling, in the presence of said target nucleicacid, amplification product; b) contacting the amplification product ofstep a) with: i. a first oligonucleotide probe which is capable ofhybridising to a first single stranded detection sequence in at leastone species within the amplification product and which is attached to amoiety that permits its detection; and ii. a second oligonucleotideprobe which is capable of hybridising to a second single strandeddetection sequence upstream or downstream of the first single strandeddetection sequence in the same strand of said at least one specieswithin the amplification product and which is attached to a solidmaterial or to a moiety that permits its attachment to a solid material;where hybridisation of the first and second probes to said at least onespecies within the amplification product produces a detector species;and c) detecting the presence of the detector species produced in stepb) wherein the presence of the detector species indicates the presenceof the target nucleic acid in said sample.
 2. A method according toclaim 1 wherein one of the first and second oligonucleotide probes isblocked at the 3′ end from extension by the DNA polymerase and is notcapable of being cleaved by either the first or second restrictionenzymes.
 3. A method according to claim 2 wherein the blockedoligonucleotide probe is rendered not capable of being cleaved by eitherthe first or second restriction enzymes due to the presence of one ormore sequence mismatch and/or one or more modifications such as aphosphorothioate linkage.
 4. A method according to claim 2 wherein theblocked oligonucleotide probe is contacted with the samplesimultaneously to the performance of step a).
 5. A method according toclaim 2 to wherein the blocked oligonucleotide probe comprises anadditional region such that the 3′ end of the species within theamplification product to which the blocked oligonucleotide probehybridises can be extended by the strand displacement DNA polymerase. 6.A method according to claim 1 wherein the sample additionally iscontacted in step a) with: (A) a third oligonucleotide primer whichthird primer comprises in the 5′ to 3′ direction one strand of therecognition sequence and cleavage site for the first restriction enzymeand a region that is capable of hybridising to the first hybridisationsequence in the target nucleic acid and wherein said third primer isblocked at the 3′ end from extension by the DNA polymerase; and/or (B) afourth oligonucleotide primer which fourth primer comprises in the 5′ to3′ direction one strand of the recognition sequence and cleavage sitefor the second restriction enzyme and a region that is capable ofhybridising to the reverse complement of the second hybridisationsequence in the target nucleic acid and wherein said fourth primer isblocked at the 3′ end from extension by the DNA polymerase. 7.(canceled)
 8. A method according to claim 1 wherein the one or moremodified dNTP is an alpha thiol modified dNTP.
 9. A method according toclaim 1 wherein the first and second restriction enzyme are the samerestriction enzyme.
 10. (canceled)
 11. A method according to claim 1wherein step a) is performed at a temperature of not more than 50° C.12. (canceled)
 13. A method according to claim 1 wherein the moiety thatpermits the detection of the first oligonucleotide probe is acolorimetric or fluorometric dye or a moiety that is capable ofattachment to a colorimetric or fluorometric dye.
 14. A method accordingto claim 1 wherein the detector species is detected by a change inelectrical signal.
 15. A method according to claim 1 wherein the moietythat permits the detection of the first oligonucleotide probe is anenzyme that yields a detectable signal following contact with asubstrate.
 16. A method according to claim 1 wherein the moiety thatpermits the attachment of the second oligonucleotide probe to a solidmaterial is a single stranded oligonucleotide.
 17. (canceled)
 18. Amethod according to claim 1 wherein in step c) the presence of thedetector species is detected by nucleic acid lateral flow.
 19. A methodaccording to claim 18 wherein the nucleic acid lateral flow utilises oneor more nucleic acids that is capable of sequence specific hybridisationto the moiety that permits the attachment of the second oligonucleotideprobe to a solid material.
 20. A method according to claim 1 whereinstep c) produces a colorimetric or electrochemical signal using carbonor gold.
 21. A method according to claim 1 wherein the first and/orsecond oligonucleotide primers comprise a stabilising sequence upstreamof the restriction enzyme recognition sequence and cleavage site.
 22. Amethod according to claim 1 wherein the hybridising region of the firstand/or second oligonucleotide primers is between 9 and 16 bases inlength.
 23. (canceled)
 24. A method according to claim 1 wherein thefirst and second hybridisation sequences in the target nucleic acid areseparated by 0 to 15 bases.
 25. A method according to claim 1 whereinthe first and second hybridisation sequences in the target nucleic acidare separated by 3 to 15 bases.
 26. A method according to claim 1wherein in step b) either the first or second single stranded detectionsequence in the at least one species within the amplification productincludes at least 3 bases of the sequence corresponding to the 3 to 15bases defined in claim
 24. 27. (canceled)
 28. A method according toclaim 1 wherein the target nucleic acid is selected from the groupconsisting of single stranded RNA, including single stranded RNA derivedfrom double stranded RNA and single stranded RNA derived from doublestranded DNA, and single stranded DNA, including single stranded DNAderived from single stranded RNA and single stranded DNA derived fromdouble stranded DNA including single stranded DNA derived from doublestranded DNA by strand invasion.
 29. (canceled)
 30. A method accordingto claim 28 wherein said single stranded DNA is derived from doublestranded DNA by use of a nuclease or derived from single stranded RNA byuse of reverse transcriptase.
 31. A method according to claim 1 whereinthe presence of two or more different target nucleic acids of definedsequence are detected in the same sample.
 32. A method according toclaim 1 wherein the sample is selected from the group consisting of anasal or nasopharyngeal swab or aspirate, blood or a sample derived fromblood, and urine.
 33. A method according to claim 1 wherein the targetnucleic acid is viral or derived from viral nucleic acid material, isbacterial or derived from bacterial nucleic acid material, iscirculating, cell-free DNA released from cancer cells or foetal cells,is micro RNA or derived from micro RNA.
 34. A method according to claim1 wherein the detection of the target nucleic acid is used for thediagnosis, prognosis or monitoring of a disease or a diseased state. 35.A method according to claim 34 wherein said disease is selected from thegroup consisting of an infectious disease, including humanimmunodeficiency virus (HIV), influenza, respiratory syncytial virus(RSV), Rhinovirus, norovirus, tuberculosis, human papillomavirus (HPV),meningitis, hepatitis, methicillin-resistant Staphylococcus aureus(MRSA), Ebola, Clostridium difficile, Epstein-Barr virus, malaria,plague, polio, chlamydia, herpes, gonorrhoea, measles, mumps, rubella,cholera or smallpox; and a cancer, including colorectal cancer, lungcancer, breast cancer, pancreatic cancer, prostate cancer, liver cancer,bladder cancer, leukaemia, esophageal cancer, ovarian cancer, kidneycancer, stomach cancer or melanoma. 36-49. (canceled)